α1 and α2-adrenergic control of large and small coronary arteries during exercise in conscious dogs under β-blockade

Summary— The aim of this study was to determine the relative roles of α₁-and α₂-adrenoceptors at the level of large epicardial and small resistance coronary arteries when sympathetic tone is increased by exercise in conscious dogs. The responses of left circumflex coronary artery diameter and blood flow were investigated at rest and during graded treadmill exercise (5, 10 and 12 km/h) in six chronically instrumented dogs during control conditions (saline) and after administration of propranolol (1 mg/kg) either alone or in combination with either prazosin (50 μg/kg), or idazoxan (300 μg/kg), or the association of prazosin + idazoxan (same doses). In control conditions, graded treadmill exercise resulted in a progressive increase in coronary artery diameter (+ 3.8 ± 0.6% from 3479 ± 80 μm) and in a decrease in coronary vascular resistance (- 46.0 ± 4.5% from 8.49 ± 1.51 mmHg/cm/s). Propranolol significantly constricted large (- 4.4 ± 0.6% from 3486 ± 87 μm) and limited dilation of small coronary arteries during exercise. These coronary effects of propranolol remained unchanged following additional α₂-adrenoceptor blockade by idazoxan but were abolished following α₁-adrenoceptor blockade by prazosin, given either alone or combined with idazoxan. Thus, α₁- but not α₂-adrenoceptors are responsible for propranolol-induced constriction of large coronary arteries and limitation of small coronary arteries dilation during exercise in conscious dogs.     

Radioimmunoassay of Prostaglandins Fα, E1 and E2 in Human Plasma

Abstract. Antibodies against prostaglandins (PG)F₂α, E₁ and E₂ were obtained in rabbits immunized with respectively PG F₂α, PG E₁ and PG E₂ conjugated to bovine serum albumin by carbodiimide. A radioimmunoassay capable of measuring 7 pg of PG Fα, 2 pg of PG E₂ and 14 pg of PG Ej in human peripheral plasma is described. Plasma samples (pH 3, citric acid) are extracted with cyclohexane: ethyl acetate, 1:1 and then chromatographed on silicic acid columns to separate the prostaglandins into three fractions: fraction I, PG A, PG B and some unknown immunoraactive compounds; fraction II, PG E and fraction III PG Fα. The recovery is 80 %± 6. 2. Mean plasma levels iu adults of PG Fa and PG E, expressed in pg/ml: -PG Fα 12 ± 2. 8 (n = 25 men), 8 ± 2. 3 (n = 18 women, follicular phase), 7 ± 1. 4 (n = 18 women, luteal phase). -PG E₁ 40. 5 + 7. 6 (n = 13 men), 38 + 17. 1 (n = 10 women). -PG E₂ 4. 5 ± 1 (n = 12 adult subjects).
The major characteristics of the method described herein are the following: - a large volume of plasma has to be processed (10 ml or more for PG Fa and PG E₁, 5 ml or more for PG E₂). - a chromatographic step is necessary to separate the different prostaglandins which makes it possible to circumvent problems of immunological cross reactivity and interference with unknown immunoreactive compounds. - great care has been taken in collection of blood samples, especially to insure complete removal of blood cells namely platelets.     

The relationship of serum total sialic acid with serum acute phase proteins and lipoprotein (a) in patients with severe hypertriglyceridaemia

Abstract. Total serum sialic acid (TSA), recently shown to be a cardiovascular risk factor, was measured in 15 patients with severe hypertriglyceridaemia (fasting triglyceride > 2·3 mmol l^-1) and 15 age and sex matched normal control subjects.
To test the hypothesis that serum TSA is related in some way to serum acute phase proteins we also measured five acute phase proteins, namely alpha-1-antichymotrypsin (ACT), alpha-1-acid-glycoprotein (AGP), alpha-2-macroglobulin (AMG), C-reactive protein (CRP) and haptoglobin (HAP) in both groups.
Of note was the significantly elevated serum TSA in the severely hypertriglyceridaemic group as compared to normal subjects. Serum TSA being 71·9 ± 11·7 mg dl^-1 and 59·6 ± 10·2 mg dl^-1 respectively ( P < 0·01 Mann-Whitney test).
Serum CRP was significantly elevated in the type IV patients as compared to controls (6·4 ± 4·5 mg l^-1 vs. 3·3 ± 1·9 mg l^-1 P <0·05 Mann Whitney test) as was serum AMG (2·1 ± 0·89 g l^-1 vs. 1·5 ± 0·53 g l^-1 P < 0·05 Mann Whitney test).
There was no correlation between serum TSA and lipoprotein (a) in either the normal or severely hypertriglyceridaemic subjects. We suggest that serum TSA could in part be related to hypertriglyceridaemia and serum acute phase proteins but that its property as a cardiovascular risk factor is not related to serum lipoprotein (a) concentrations.     

Thromboxane A2 and related prostaglandins in airways

Summary— Asthma is now thought to be a chronic inflammatory disease of the airways. The roles of prostanoids, thromboxane A₂ (TXA₂) and the prostaglandins (PGs) in the pathogenesis and pathophysiology of asthma have fostered a wealth of studies but remain controversial. TXA₂ and the bronchoconstrictor PGs, PGD₂ and PGF_2α, are generated in greater amounts in asthmatic than in normal subjects. TXA₂ is a potent constrictor of airway smooth muscle, an inducer of acetylcholine release and of airway microvascular leakage. It may participate in the thickening and the remodeling of the airway wall which may contribute to the airway hyperresponsiveness, a typical feature of asthma. Strategies for inhibition of TXA₂ effects include antagonism of the TXA₂ receptor (TP receptor) and inhibition of the thromboxane synthase. TP receptor antagonists could block the effects of all the bronchoconstrictor prostanoids because TXA₂ as well as the bronchoconstrictor PGs act through activation of lung TP receptor. The recent development of specific and potent TP receptor antagonists and inhibitors of thromboxane synthase has provided tools to assess the role of TXA₂ and bronchoconstrictor PGs in the pathophysiology of asthma.     

Urinary excretion of bile alcohols in normal children and patients with α1-antitrypsin deficiency during development of liver disease

Abstract. Healthy infants and children were found to excrete bile alcohol glucuronides in urine. Following isolation and hydrolysis, the bile alcohols were estimated by capillary gas-liquid chromatography. The daily urinary excretion of the major compound, 27-nor-5 β -cholestane-3α,7α,12α,24ξ,25ξ-pentol (a C₂₆ bile alcohol), ranged from 0·1 to 1·1 μmol/24 h per m² body surface area for healthy infants and children. Two groups of patients with α₁-antitrypsin deficiency (phenotype PiZ) were also studied during infancy and childhood, and biochemical liver function tests and liver morphology were compared to the excretion of bile alcohols. The highest excretion of the C₂₆ bile alcohol in urine was found in patients with α₁-antitrypsin deficiency and juvenile cirrhosis (2·1–8·4 μmol 24 h^-1 m^-2) regardless of preceding neonatal cholestasis. Patients with α₁-antitrypsin deficiency, neonatal cholestasis and subsequent fibrosis or normal liver morphology excreted bile alcohols within the normal range. The C₂₆ bile alochol constituted an average of 36% of the total bile alcohols in forty-three urine samples. This percentage was about the same in the three groups studied. The findings suggest that determination of urinary bile alcohols may be a valuable non-invasive diagnostic tool for patients with or at risk of developing liver cirrhosis.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Effects of high-altitude chronic hypoxia on platelet α2-receptors in man

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike β-adrenergic receptors, to our knowledge no data are available on α-receptors. We studied platelet α₂- and leucocyte β-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet α₂-receptor density and affinity [ B _max from 92.6 ± 6.7 to 54.6 ± 4.2 fmol mg⁻¹ protein ( P  < 0.001) and K _D from 1.271 ± 0.034 to 1.724 ± 0.077 nmol L⁻¹ ( P  < 0.05)], although no changes to β-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of α₂-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.     

Effect of vitamin B6 administration on elevated plasma oxalic acid levels in haemodialysed patients

Abstract. Accumulation of oxalic acid resulting in elevated plasma levels is a common finding in uraemic patients. Since vitamin B₆ is an important coenzyme in oxalic acid metabolism the influence of vitamin B₆ administration on plasma oxalic acid levels was investigated.
Vitamin B₆ was administered to eight chronic haemodialysis patients with secondary hyperoxalaemia. Mean plasma oxalic acid concentration decreased from 149·5 ± 67 μmol/l to 99·0 ± 36·4 μmol/l within 2 weeks and to 93·8 ± 33·1 μmol/l after 4 weeks of pyridoxine treatment ( P <0·01) the mean reduction being 46% (32·0–56·1%). The decrease in plasma oxalic acid levels was most pronounced in patients with the highest pretreatment values. Two patients who received pyridoxine therapy prior to the beginning of the study had low initial values of plasma oxalic acid concentrations and showed no further decline.     

Direct determination of leucine metabolism and protein breakdown in humans using L-[1-13C, 15N]-leucine and the forearm model

Abstract. We have used the forearm model to study protein metabolism in six normal healthy subjects in the fed state using L-[1 –¹³C, ¹⁵N]-leucine as the substrate tracer.
Deep venous and arterialized venous blood samples from the forearm were collected at 10-min intervals 2±5 h into a primed-continuous infusion of the dilabelled tracer. Arterialized venous blood was obtained using a 'hot-box' technique and forearm blood flow was measured by mercury strain-gauge plethysmography.
The concentration and isotope enrichment of leucine and its metabolites, α-ketoisocaproic acid and CO₂, in deep venous and arterialized venous blood were measured by gas chromatography-mass spectrometry and isotope ratio-mass spectrometry.
The rates of leucine deamination and reamination were 388 ± 24 (mean ± SEM) and 330 ± 23 nmol (100 ml)^-1 min^-1 respectively, whilst protein synthesis and breakdown rates were 127 ± 11 and 87 ± 10 nmol (100 ml)^-1 min^-1 respectively across the forearm in the fed state. We have demonstrated that the use of doubly labelled leucine as tracer and application of the mathematical model developed in this study, permits the comprehensive quantification of leucine kinetics including protein breakdown.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

Familial benign hypercalcaemia: a possible abnormality in calcium transport by erythrocytes

Abstract. The fundamental biochemical abnormality in familial benign hypercalcaemia (FBH) (familial hypocalciuric hypercalcaemia) is unknown. It seemed possible that, since the kidneys and the parathyroid glands are insensitive to the high extracellular calcium levels, a general disorder of the regulation of the calcium pump on the plasma membrane is present. We obtained evidence suggesting that active calcium efflux by erythrocytes from patients with FBH (85·7 pL 4·5 μmol l^-1 min^-1) is higher ( P < 0·005) than that by erythrocytes from control subjects (78·6 pL 4·1 μmol l^-1 min^-1) or from patients with primary hyperparathyroidism (77·5 pL 5·2 μmol l^-1 min^-1, P < 0·05). Calcium influx into erythrocytes was normal in FBH and in primary hyperparathyroidism.     

CHANGES IN PLATELET FREE Ca2+ CONCENTRATION AFTER CHRONIC DIGOXIN TREATMENT

Summary— Cell Na+ and Ca²⁺ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca²⁺ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca²⁺-countertransport system. Cytosolic free Ca²⁺ concentration is also controlled by the Na+–H+ exchange activity. To analyze the changes in cytosolic Ca²⁺ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca²⁺]_i was increased in platelets from 169±30 to 321±61 nmol/l ( n = 7, P <0.02) and unchanged in erythrocytes (121±6 and 104±7 nmol/l). This increase in platelet [Ca²⁺]_i was not accompanied by a change in serotonin content (5.43±0.67 vs 5.49±0.61 10⁻⁷ mol per 10¹¹ cells) and could not be reproduced by in vitro addition of 10⁻⁴ mol/l ouabain (198±33 vs 186±73 nmol/l). The enhanced [Ca²⁺]_i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca²⁺ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.     

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery

Summary— The involvement of large conductance Ca²⁺-activated K⁺ channels (BK) and ATP-sensitive K⁺ (K_ATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca²⁺]_i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca²⁺]_i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca²⁺]_i with force in the presence of serotonin at different extracellular Ca²⁺ concentration ([Ca²⁺]_o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca²⁺-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.     

Increased production of platelet thromboxane B2 in non-insulin-dependent diabetes. Relationship to vascular complications

Abstract. Platelet thromboxane B₂ production was studied in forty-seven non-insulin-dependent diabetics by incubating platelets with increasing concentrations of arachidonic acid. In comparison with thirty-two healthy subjects, diabetics showed increased thromboxane B₂ production at 0·7 mmol/l (mean: 236 pmol/10⁸ platelets, SEM 201–277; v. 135, 105–174; P < 0·05) and at 1·0 mmol/l (673, 613–739; v. 405, 377–486, P < 0·01) but not at 0·5 mmol/l. Patients were subdivided according to the presence or absence of vascular complications. Patients without microangiopathy showed significantly greater thromboxane B₂ production than healthy subjects at all the arachidonic-acid concentrations ( P < 0·02 or less). Patients with microangiopathy had platelet thromboxane production similar to that observed in healthy subjects at all the arachidonic-acid concentrations ( P > 0·30) but significantly lower than that of non-microangiopathic patients at 0·5 ( P < 0·01) and at 0·7 mmol/l arachidonic acid ( P < 0·05). These results indicate that non-insulin-dependent diabetics have increased production of platelet thromboxane B₂ only when they do not have microvascular complications.     

Prostaglandin E2 (PGE2) induces headache in healthy subjects

The role of prostanoids in nociception is well established. The headache-eliciting effects of prostaglandin E₂ (PGE₂) and its possible mechanisms have previously not been systematically studied in man. We hypothesized that infusion of PGE₂ might induce headache and vasodilation of cranial vessels. PGE₂ (0.40 µg kg⁻¹ min⁻¹) or saline was infused for 25 min into 11 healthy subjects in a cross-over, double-blind study. Headache intensity was scored on a verbal rating scale from 0 to 10. In addition, we recorded mean flow in the middle cerebral artery (V_MCA) by transcranial Doppler and diameter of the superficial temporal artery (STA) by high-resolution ultrasonography. All 11 subjects reported headache on the PGE₂ day and no subjects reported headache on the placebo day ( P  = 0.001). During the immediate phase (0–30 min) ( P  = 0.005) and the postinfusion phase (30–90 min) ( P  = 0.005), the area under the curve for headache score was significantly larger on the PGE₂ day compared with the placebo day. PGE₂ caused dilatation of the STA (23.5%; 95% CI 14.0, 37.8) and the MCA (8.3%; 95% CI 4.0, 12.6). We suggest that PGE₂ induces headache by activation and sensitization of cranial perivascular sensory afferents.     

Potentiation of platelet activation through the stimulation of P2X1 receptors

Summary.  The platelet P2X₁ purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X₁ in platelet function is unknown, though stimulation with the P2X₁ agonist α,β-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X₁ stimulation on platelet activation. Stimulation of P2X₁ with α,β-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to α,β-Me-ATP alone was less than that observed in heparin; however, α,β-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X₁ antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by α,β-Me-ATP. Overall, this study demonstrates that P2X₁ stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X₁ stimulation, supporting a role for this receptor in regulating platelet activation.     

Protoporphyrinogen oxidase and porphobilinogen deaminase in variegate porphyria

Abstract. Two enzymes of the haem biosynthetic pathway were investigated in patients with variegate porphyria. Protoporphyrinogen oxidase in cultures of Epstein-Barr virus transformed lymphoblasts from twenty-seven patients showed a mean maximal velocity ( V _max) of 0·39 ± 0·08⁺ nmol of protoporphyrin mg protein^-1 h^-1, a 52% reduction ( P < 0·001) from a non-porphyric control group (0·82 ± 0·10). K _m values (1·00 ± 0·27 μ M) did not differ significantly ( P > 0·05) from control values in any of the patients. The mean V _max of porphobilinogen deaminase in the cultures was 1·50 ± 0·18 nmol of uroporphyrin mg protein^-1 min^-1, a 24% reduction ( P < 0·001) from controls (1·94 ± 0·14). Mean porphobilinogen deaminase activity in the erythrocytes of twenty-one patients with variegate porphyria was 8·37 ± 1·99 nmol of uroporphyrin 1 erythrocytes^-1 s^-1, a 28% reduction ( P < 0·001) from normal (11·98 ± 2·11). The reduced activities of these two enzymes comply with the expression of variegate porphyria during its quiescent and acute phases.     

The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Cisplatin-induced renal effects and thromboxane A2 receptor blockade

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A₂ receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 ± 25 mL min⁻¹ ( P  = 0.049 vs. baseline) and effective renal plasma flow by 94 ± 150 mL min⁻¹ ( P  = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 ± 18 mL min⁻¹ ( P  = 0.050 vs. baseline) and in effective renal plasma flow by 26 ± 63 mL min⁻¹ ( P  = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.