Deleterious effect of glycation on the ability of HDL to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation.

BACKGROUND: Contrary to high-density lipoprotein (HDL) from normolipidaemic and normoglycaemic subjects, HDL from diabetic patients loses its ability to reverse the inhibition of vasorelaxation induced by oxidized low-density lipoprotein (LDL). The aim of this study was to analyze the role of glycation, a major abnormality observed in diabetes, on the impairment of the vasorelaxant effect of HDL. METHODS: HDL from healthy subjects was glycated in vitro by incubation in glucose 200 mmol/L for 3 days. Vasoreactivity was evaluated by the relaxation response to acetylcholine of rabbit aorta rings pre-contracted with noradrenaline, before and after 2 h incubation with or without different lipoprotein fractions (Krebs buffer, oxidized LDL, normal or glycated HDL alone and with oxidized LDL). RESULT: The fructosamine/apolipoprotein AI ratio was significantly increased in glycated HDL compared with native HDL (53.63 ± 7.91 vs 18.51 ± 4.10 µmol/g; p < 0.05). Oxidized LDL inhibited endothelium-dependent vasodilation compared with Krebs buffer [maximal relaxation (Emax) = 53.15 ± 6.50 vs 98.67 ± 2.07%, p < 0.001]. Native HDL was able to counteract the oxidized LDL-induced inhibition of vasorelaxation (Emax = 76.93 ± 5.41 vs 53.15 ± 6.50%, p < 0.001). On the other hand, glycated HDL had no effect on oxidized LDL-induced inhibition of endothelium vasorelaxation compared with incubation with oxidized LDL alone (Emax = 52.98 ± 2.07 vs 53.15 ± 6.50%, not significant). CONCLUSION: Glycation of HDL induces the loss of the ability of HDL to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation, this is likely contributing to the impairment of antiatherogenic properties of HDL in diabetic patients.

HDL particles from type 1 diabetic patients are unable to reverse the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation.

AIMS/HYPOTHESIS: In healthy individuals, HDL can counteract the inhibition of vasorelaxation induced by oxidised LDL. Several abnormalities such as increased size, glycation and decreased paraoxonase activity have been reported for HDL from type 1 diabetic patients. Thus, we hypothesised that the ability of HDL to protect vessels against impairments of vasorelaxation would be decreased in these patients. METHODS: We compared the ability of HDL from 18 type 1 diabetic patients and 12 control participants to counteract the inhibition of endothelium-dependent relaxation induced by oxidised LDL on rabbit aorta rings. RESULTS: Serum triacylglycerol and total cholesterol, LDL- and HDL-cholesterol were similar in type 1 diabetic and control participants. Fasting glycaemia and the HDL-fructosamine level were higher in diabetic patients than in controls (9.06 +/- 3.55 vs 5.27 +/- 0.23 mmol/l, p < 0.005; and 10.2 +/- 3.2 vs 7.7 +/- 2.5 micromol/g protein, p < 0.05, respectively). HDL composition, size and paraoxonase activity were similar in both groups. HDL from controls reduced the inhibitory effect of oxidised LDL on maximal relaxation (E (max); 79.3 +/- 11.8 vs 66.4 +/- 11.7%, p < 0.05), whereas HDL from type 1 diabetic patients had no effect (E (max) = 70.6 +/- 17.4 vs 63.9 +/- 17.2%, NS). In type 1 diabetic patients, E (max) was not correlated with glycaemia or the HDL-fructosamine level. CONCLUSIONS/INTERPRETATION: HDL particles from type 1 diabetic patients do not protect against inhibition of endothelium-dependent vasorelaxation induced by oxidised LDL, in contrast to HDL particles from healthy individuals. This defect cannot be explained by abnormalities in HDL composition, size or paraoxonase activity, and may contribute to the early development of atherosclerotic lesions in type 1 diabetic patients.

Inability of HDL from type 2 diabetic patients to counteract the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation.

AIMS/HYPOTHESIS: In healthy normolipidaemic and normoglycaemic control subjects, HDL are able to reverse the inhibition of vasodilation that is induced by oxidised LDL. In type 2 diabetic patients, HDL are glycated and more triglyceride-rich than in control subjects. These alterations are likely to modify the capacity of HDL to reverse the inhibition of vasodilation induced by oxidised LDL. SUBJECTS AND METHODS: Using rabbit aorta rings, we compared the ability of HDL from 16 type 2 diabetic patients and 13 control subjects to suppress the inhibition of vasodilation that is induced by oxidised LDL. RESULTS: Oxidised LDL inhibited endothelium-dependent vasodilation (maximal relaxation [Emax] = 58.2+/-14.6 vs 99.3+/-5.2% for incubation without any lipoprotein, p < 0.0001). HDL from control subjects significantly reduced the inhibitory effect of oxidised LDL on vasodilatation (Emax = 77.6+/-12.9 vs 59.5+/-7.7%, p < 0.001), whereas HDL from type 2 diabetic patients had no effect (Emax = 52.4+/-20.4 vs 57.2+/-18.7%, NS). HDL triglyceride content was significantly higher in type 2 diabetic patients than in control subjects (5.3+/-2.2 vs 3.1+/-1.4%, p < 0.01) and was highly inversely correlated to Emax for oxidised LDL+HDL in type 2 diabetic patients (r = -0.71, p < 0.005). CONCLUSIONS/INTERPRETATION: In type 2 diabetes mellitus, the ability of HDL to counteract the inhibition of endothelium-dependent vasorelaxation induced by oxidised LDL is impaired and is inversely correlated with HDL triglyceride content. These findings suggest that HDL are less atheroprotective in type 2 diabetic patients than in control subjects.

Co-incubation of native and oxidized low-density lipoproteins: potentiation of relaxation impairment.

The influence of native low-density lipoprotein (LDL) on the inhibition of endothelium-dependent relaxation previously induced by oxidized LDL was investigated with intact rabbit aortic rings. We also tried to assess oxysterol involvement in the native lipoprotein effects. Lipoprotein fractions (1 mg protein/ml) were tested for their ability to inhibit the vasorelaxation induced by acetylcholine in aorta rings previously precontracted by noradrenaline vs. that in control strips in Krebs buffer. Co-incubation of oxidized and native LDL reinforced the oxidized LDL-induced inhibition, compared to the impairment evoked by oxidized LDL alone (E(max)=43.3+/-6.7% and 61. 4+/-5.4%, respectively; P<0.05). Finally, smaller amounts of 7-oxy-cholesterols were recovered in organ baths after co-incubation of native and oxidized LDL than after incubation of oxidized LDL alone. Conversely, more oxy-cholesterols were found in the strip vessels under the same conditions (% of oxysterol incorporation: 0. 05158 vs. 0.10199, r=0.703). Together these results suggest that the strengthening of oxidized LDL-induced inhibition by native LDL is dependent on an oxysterol effect on arterial wall cells. Mechanisms involved in this phenomenon remain to be investigated.

Mass concentration of plasma phospholipid transfer protein in normolipidemic, type IIa hyperlipidemic, type IIb hyperlipidemic, and non-insulin-dependent diabetic subjects as measured by a specific ELISA.

Mean plasma phospholipid transfer protein (PLTP) concentrations were measured for the first time by using a competitive enzyme-linked immunosorbent assay. PLTP mass levels and phospholipid transfer activity values, which were significantly correlated among normolipidemic plasma samples (r=0.787, P<0.0001), did not differ between normolipidemic subjects (3.95+/-1.04 mg/L and 575+/-81 nmol. mL-1. h-1, respectively; n=30), type IIa hyperlipidemic patients (4. 06+/-0.84 mg/L and 571+/-43 nmol. mL-1. h-1, respectively; n=36), and type IIb hyperlipidemic patients (3.90+/-0.79 mg/L and 575+/-48 nmol. mL-1. h-1, respectively; n=33). No significant correlations with plasma lipid parameters were observed among the various study groups. In contrast, plasma concentrations of the related cholesteryl ester transfer protein (CETP) were higher in type IIa and type IIb patients than in normolipidemic controls, and significant, positive correlations with total and low density lipoprotein cholesterol levels were noted. Interestingly, plasma PLTP mass concentration and plasma phospholipid transfer activity were significantly higher in patients with non-insulin-dependent diabetes mellitus (n=50) than in normolipidemic controls (6.76+/-1. 93 versus 3.95+/-1.04 mg/L, P<0.0001; and 685+/-75 versus 575+/-81 nmol. mL-1. h-1, P<0.0001, respectively). In contrast, CETP levels did not differ significantly between the 2 groups. Among non-insulin-dependent diabetes mellitus patients, PLTP levels were positively correlated with fasting glycemia and glycohemoglobin levels (r=0.341, P=0.0220; and r=0.382, P=0.0097, respectively) but not with plasma lipid parameters. It is proposed that plasma PLTP mass levels are related to glucose metabolism rather than to lipid metabolism.

Isotope ratio mass spectrometry, compared with conventional mass spectrometry in kinetic studies at low and high enrichment levels: application to lipoprotein kinetics.

The aim of the present study was to compare the performances of gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in stable isotope kinetic studies. In the analysis of cholesterol and leucine, GC/C/IRMS gave precise and linear results over a large scale of 13C enrichment (-22 to +760 delta/1000 for cholesterol, -26 to +600 delta/1000 for leucine). Compared with GC/MS, GC/C/IRMS was much more accurate and reproducible, especially at low [13C]-cholesterol enrichment (-12 delta/1000), with cholesterol samples ranging from 0.11 to 17 ng. Cholesterol ester kinetics in rabbit plasma low-density lipoproteins was studied after injection of 3 mg [3,4-(13)C]cholesterol. A smooth and regular kinetic curve was obtained with GC/C/IRMS; results were much less reproducible with GC/MS. Finally, the performances of GC/C/IRMS were demonstrated in the simultaneous kinetic study of three human plasma apolipoproteins during a primed constant infusion of 0.7 mg.kg-1.h-1 L-[l-13C]leucine. Kinetic curves were obtained in very-low-density lipoproteins and low-density lipoproteins for apolipoprotein B100, and in high-density lipoproteins for apolipoproteins AI and AIL.

Inhibitors of arterial relaxation among components of human oxidized low-density lipoproteins. Cholesterol derivatives oxidized in position 7 are potent inhibitors of endothelium-dependent relaxation.

BACKGROUND: Oxidized low-density lipoproteins (LDLs) are known to impair arterial relaxation. The aim of the present study was to identify the components of oxidized LDL that may account for inhibition of endothelium-dependent relaxation. METHODS AND RESULTS: LDLs from 12 healthy subjects were either maintained at 4 degrees C (native LDL) or incubated at 37 degrees C in the presence of copper sulfate (oxidized LDL). Unlike pretreatment with native LDL, pretreatment with oxidized LDL reduced significantly the acetylcholine-mediated relaxation of rabbit aortic segments compared with control segments incubated in Krebs' buffer (maximal relaxation [Emax], 72.0 +/- 6.7% versus 94.1 +/- 0.8%, respectively, P < .01; negative logarithm of the concentration required to produce a half-maximal relaxing effect [pD2], 6.6 +/- 0.1 versus 7.2 +/- 0.1, respectively, P < .001). The absolute difference between Emax values obtained with oxidized and native LDL (delta Emax) correlated significantly with the formation of 7 ketocholesterol, 7 alpha-hydroxycholesterol, and 7 beta-hydroxy-cholesterol. In contrast, delta Emax did not correlate with the amount of lipoperoxides or lysophosphatidylcholine formed, and the difference of pD2 values measured with oxidized and native LDL (delta pD2) did not correlate significantly with any of the oxidation-derived LDL compounds. When added individually, 7-ketocholesterol and 7 beta-hydroxycholesterol reduced Emax values but not pD2 values in a time- and concentration-dependent manner. CONCLUSIONS: Cholesterol derivatives in oxidized LDL can reduce maximal arterial relaxation through a specific effect on vascular endothelial cells.

Capillary gel electrophoresis analysis of apolipoproteins A-I and A-II in human high-density lipoproteins.

Capillary gel electrophoresis was performed on a coated capillary column filled with a replaceable low-viscosity polymer network containing sodium dodecyl sulfate (eCAP SDS-200 kit from Beckman Instruments). While apolipoprotein (apo) A-I gave an homogeneous peak in high-density lipoprotein (HDL), it appeared heterogeneous in its purified form. Apo A-II was heterogeneous in HDL as well as in purified preparations. For both proteins the relationship between peak areas and apo concentrations was linear over a large range of concentrations and, with the use of alpha-chymotrypsinogen A as an internal standard, apo A-I and apo A-II areas were measured with good precision (coefficient of variation 1.6 and 1.8%, respectively). Capillary gel electrophoresis appeared as a method of high-resolution power in the study of apo A-I and apo A-II heterogeneity and could lead to the development of an alternative method for the assay of apo HDL.

Influence of apolipoprotein composition of high density lipoprotein particles on cholesteryl ester transfer protein activity. Particles containing various proportions of apolipoproteins AI and AII.

The effect of apolipoprotein (apo) composition of high density lipoproteins (HDL) on cholesteryl ester transfer protein (CETP) activity was studied by measuring the rate of radiolabeled cholesteryl esters transferred between low density lipoproteins (LDL) and HDL3 which contained various proportions of apoAI and apoAII. Ultracentrifugally isolated HDL3, which contained virtually only apoAI and apoAII in their protein moiety, were progressively enriched with apoAII upon the incubation with increasing amounts of delipidated HDL apolipoproteins. The substitution of apoAII for apoAI in HDL3 did not induce marked alteration of the lipid composition of the lipoprotein particles. The rates of cholesteryl ester exchanges with LDL in the presence of purified human CETP were significantly reduced with apoAII-enriched HDL3 as compared with non-enriched homologous particles. Consistent results were obtained by determining the rate of cholesteryl esters transferred either from LDL toward HDL3, or in the opposite direction, from HDL3 to LDL. The effect of the apoAI and apoAII content of HDL particles on CETP activity was also investigated by measuring the rate of cholesteryl esters transferred from LDL to plasma HDL3 particles which contained either only apoAI, HDL3-AI, or both apoAI and apoAII, HDL3-AIAII. HDL3-AI and HDL3-AIAII particles were isolated from human plasma by a sequential procedure which combined ultracentrifugation and anti-apoAII immunoaffinity chromatography. As observed with HDL3 artificially enriched with apoAII, cholesteryl ester transfer rates were significantly lower with plasma HDL3-AIAII than with plasma HDL3-AI particles. Kinetic analysis of the interaction of CETP with apoAII-enriched HDL3 revealed that apoAII could act as an uncompetitive inhibitor of the cholesteryl ester transfer reaction. Since the plasma levels of HDL-AI, HDL-AIAII, and HDL-AII may undergo significant physiological fluctuation, the present study suggests that HDL apoproteins may be important factors in modulating cholesteryl ester transfer rates in vivo.

Characteristics of slow bursting activities recorded in cervical ventral roots in the in vitro brainstem-spinal cord preparation of the neonatal rat.

The aim of the present work was to disclose, through pharmacological activation of an isolated central nervous system maintained in vitro, spinal locomotor and respiratory-like activities inferred from an in vivo rabbit preparation. In a brainstem-spinal cord preparation in neonatal rats (0-3 days old), medullary respiratory activity occurred spontaneously in the cervical ventral roots. During 5-hydroxytryptophan (5-HTP) superfusion (0.2 mM), a slower rhythm with longer burst duration developed in the same ventral roots, with the pre-existing long-lasting slow bursting (LLSB) activity. At the same time, locomotor bursts were recorded from lumbar ventral roots. The LLSB activity was mainly recorded in cervical ventral roots, but they could also be encountered at the lumbar level, where they were eliminated after thoracic transection. The LLSB activity and the locomotor bursting were maintained after a C1 or C2 spinal transection, whereas medullary activity disappeared. Bilateral recording of the three types of rhythmic activity demonstrated that the LLSB activity and the medullary respiratory bursting typically displayed a synchronous bilateral coupling, whereas at caudal levels an alternate bilateral pattern was the rule for locomotor activity. Lactic acid could reinduce LLSB activity if introduced after it had just disappeared during the washout phase following 5-HTP superfusion. These results strongly suggest that the LLSB activity that originates from cervical generators belongs to the respiratory system, and not to locomotor activity. Finally, similar results in an in vivo rabbit preparation have been obtained through pharmacological activation. This preparation appears to be a suitable model for the analysis of this cervical burst generator and for the study of interactions among the different pattern generators.

Characterization of hindlimb muscle afferents involved in ventilatory effects observed in decerebrate and spinal preparations.

Neurogenic changes of phrenic activity have previously been observed during periodic passive motions of one hindlimb in decorticate, unanaesthetized and curarized rabbit preparations before and after high spinal transection (Palisses et al. 1988). In decerebrate and spinal preparations, we aimed to determine, through rhythmic electrical stimulation of hindlimb muscle nerves, which muscle afferents are involved in these effects. In decerebrate preparations, these electrical stimulations (trains of shocks at 80 Hz for 300 ms every second for 20 s) produced ventilatory effects when group I + II afferent fibres of either flexor or extensor nerves were stimulated together and more powerful changes as soon as group III fibres were recruited. Stimulation of group I fibres alone induced no such effects. When present, these changes in respiratory activity consisted of a maintained decrease of the respiratory period due to both inspiratory and expiratory time shortening; in addition, the amplitude of the phrenic bursts greatly increased at the onset of electrical stimulation. After spinal transection at C2 level and pharmacological activation by nialamide and DOPA, only short-lasting phrenic bursts developed spontaneously; the electrical stimulation of group II and mainly group III flexor afferent fibres induced large amplitude phrenic activity whereas the stimulation of the same extensor afferents was relatively ineffective. The activation of phrenic motoneurones during group III flexor afferent stimulation was closely linked to each 300 ms period of stimulation. While the phrenic effects obtained in the spinal preparations by natural and by electrical periodic stimulation are quite similar to each other, those produced in decerebrate preparations differ substantially.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the entrainment of breathing by locomotor pattern in human.

In human, it has been shown that interactions between locomotor and respiratory patterns may lead to locomotor-respiratory couplings termed entrainment. In order to prove that this coupling is really an entrainment, we tried to show that it obeys one of the expected rules, i.e. that it evolves and is not present for all imposed locomotor frequencies. For that purpose, seventeen healthy volunteers were asked to run on a treadmill at 14 different locomotor rates (instead of 2 or 3 in previous works) for 40 s. All the subjects did not exhibit the same coupling and different relationships could be obtained: the most commonly observed was 2:1 (2 locomotor activities for a respiratory one) but other forms could appear (4:1 and even 5:2 or 3:2). When the coupling evolution was followed in the same subject, it did not appear for all locomotor frequencies but only for locomotor periods close to harmonics of respiratory ones (absolute coordination). On both sides of these values, it progressively evolved to relative coordination and to the lack of coordination. When two forms of absolute coordination were observed in a same subject, the phase relationships followed the rules of the entrainment. Compared to data obtained in quadrupeds, these results suggest that the entrainment of breathing frequency by the locomotor activity is due to central interactions between the respiratory and locomotor pattern generators and does not depend on a chemical regulation avoided here by short locomotor sequences.

Evidence for respiratory interneurones in the C3-C5 cervical spinal cord in the decorticate rabbit.

In mammals, it has long been considered that the bulbo-spinal inspiratory drive provided a direct (monosynaptic) excitation of phrenic motoneurones (Phr Mns). Although such connections have been demonstrated, recent indirect data strongly suggested that the main inspiratory drive is polysynaptic. We tried to directly demonstrate relay respiratory interneurones at the C3-C6 spinal cord level where the Phr Mn pool is located. The experiments were performed on decorticate, unanaesthetized, bilaterally vagotomized and curarized rabbits and the firing pattern of spinal interneurones was compared to the phrenic bursting. Dorsally and dorso-medially to the Phr Mn pool, different classes of inspiratory (54%) and expiratory (46%) interneurones could be identified in the ventral horn. Three classes of inspiratory interneurones were characterized and classified as "I all" (26%), "I late" (43%) and "I tonic" (29%) according to the terminology used by other authors for the bulbospinal inspiratory neurones which drive the spinal respiratory motoneurones. The expiratory interneurones could also be divided into 3 classes: "E all" (48%), "E late" (10%) and "E tonic" (41%). This first direct evidence of inspiratory interneurones at the C3-C6 spinal cord levels can account for the major polysynaptic excitation of the Phr Mns while the presence of numerous expiratory interneurones at this level suggests a polysynaptic bulbo-spinal inhibitory action onto the Phr Mns. These classes of inspiratory and expiratory interneurones did not always coincide with the bulbo-spinal classes of neurones described elsewhere.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflex modulation of phrenic activity through hindlimb passive motion in decorticate and spinal rabbit preparation.

The neurogenic effect of passive hindlimb movement on phrenic nerve discharge was compared in decorticate unanaesthetized and curarized rabbit preparations prior to and after spinal transection. The question of how and where sensory information has access to the central respiratory network was addressed in each case. All passive motions, performed using a mechanical device, were of constant amplitude in a given preparation. The results clearly differed in decorticate and spinal preparations. In the decorticate vagotomized preparation, periodic passive motions led to an immediate shortening of the respiratory period which lasted throughout the periodic stimulation and stopped with its cessation; it did not depend on the frequency of the natural stimulation and was entirely due to a 20% shortening of the expiration time. Maintained full flexion or full extension both induced the same expiration time shortening, but limited to the first two to three respiratory cycles after onset and interruption of stimulation. After spinal transection at the C2 level, and moderate activation with DOPA, no phrenic activity developed in the absence of proprioceptive stimulation. Periodic hindlimb movements evoked simultaneous large bursts in both phrenic nerves during each extension; a 1:1 coordination of phrenic activity with the external imposed period (P) was observed for various P values. A strong phrenic activation could also be elicited through maintained full hindlimb extension but not through full flexion: this activation appeared as rhythmic discharge as long as extension was maintained. It is concluded that proprioceptive inputs act upon the medullary respiration generator and reset its own rhythm whereas, at the spinal level, they elicit an amplitude modulation at phrenic motoneuronal level without acting upon the rate of the spinal "respiration" generator itself; on the same phrenic motoneurons, a subthreshold central activation added to a subthreshold proprioceptive activation probably accounts for the phrenic bursting during maintained extension. Finally, the proprioceptive control from the hindlimb on phrenic activity is processed at different sites of the central respiratory network at medullary and at spinal level, and may depend on different input signals.

Evidence for central entrainment of the medullary respiratory pattern by the locomotor pattern in the rabbit.

1) Although periodic passive hindlimb movements can reproduce the enhancement of breathing frequency seen at the onset of muscular exercise, we have shown previously that they were unable to induce the 1:1 coupling which is observed between locomotion and respiration during galloping in quadrupeds. The purpose of this study was to investigate the existence of a central coupling in two experimental situations: first, decorticate - DOPA, and secondly, decerebrate rabbit preparations. 2) After DOPA administration in curarized, vagotomized, decorticate animals, an absolute coordination could be observed between the locomotor bursts (which developed in hindlimb muscle nerves) and phrenic activity. With the temporal evolution of the pharmacological activation, the coupling mode varied from 1:1 to 1:2 during the same experiment with a loss of coordination between these two forms. When the coordination between both motor activities was not produced in such conditions, it could be induced for some imposed frequencies of periodic passive motions applied to the contralateral hindlimb. 3) When the DOPA effects were completely over, a rostro-pontine decerebration allowed locomotor activity to be released and a tight 1:1 coupling could be obtained again between the two motor patterns in this new experimental situation. 4) An analysis of the data revealed that the various forms of coordination obtained in the different experimental situations are due to a central resetting of the respiratory and of the locomotor patterns. The capability of the hindlimb proprioceptive inputs to coordinate locomotor and respiratory patterns in the decorticate-DOPA preparation appeared simply linked to their ability to entrain the activity of the lumbar locomotion generator. It is suggested that these central reciprocal interactions, which have the properties of an entrainment process, are the result of interactions between the lumbar locomotion generator and the medullary respiratory one.

Different mechanisms involved in supraspinal and spinal reflex regulation of phrenic activity through chest movements.

The coordination of breathing activity with chest movements was compared in the same decorticate rabbit preparations prior to and after a transection at the C2 spinal level. Pharmacological activation was induced with a combination of nialamide and DOPA in the latter situation. The preparation was curarized and chest inflations and deflations were induced by a respirator whose parameters could be modified. In decorticate preparations, phrenic activity was coordinated 1:1 with the respirator period over a large range of imposed periods. Beyond the extreme values a new coupling was achieved with a ratio of either 1:2 or 2:1. Throughout the range of 1:1 coordination, phrenic bursting always happened at a preferred time in the respirator period, although this time differed for the various imposed periods. This coordinated activity required vagal inputs. After spinal transection the phrenic nerves were totally silent; DOPA administration allowed rhythmic activity to develop. In some preparations, phrenic bursts were coordinated 1:1 with the respirator period and remained so for all the imposed periods: the phase of these phrenic discharges relative to the respirator cycle was kept unchanged for the different periods. In addition, there was a modulation of amplitude superimposed on this 1:1 coupling. These spinal phrenic bursts were generally suppressed when the respirator was turned off. From these results, the coordination of phrenic activity with the respirator rate appears to be produced by different mechanisms in the decorticate and in the spinal preparations. In the decorticate animal the periodic vagal inflow reset the activity of the medullary inspiratory generator and entrains it at its own rate. The coordination observed in the spinal preparation results from a periodic peripheral activation of premotoneuronal or motoneural phrenic elements during inflation. If the central bursts provided by the spinal "respiration" generator can fire phrenic motoneurons above threshold, their timing is not dependent on the peripheral inflow; when the motoneurons are fired below threshold by these central inputs, they are probably summing together the central and peripheral excitations, which could account for the amplitude modulation of the coordinated phrenic bursts of pure reflex origin. Possible afferent pathways are discussed.

Changeover from alternate to synchronous bilateral pattern of the phrenic bursts entrained by fictive locomotion in the spinal rabbit preparation.

Phrenic bursting resulting from locomotor entrainment during fictive locomotion was shown previously in high spinal preparation after nialamide-DOPA administration. The temporal evolution of the bilateral pattern of phrenic vs locomotor activity is considered here. At variance with the bilateral locomotor pattern which is always alternate (fictive stepping), the pattern on both phrenic nerves changes with time after DOPA injection: first alternate, left and right phrenic bursts become synchronous. A study of ipsilateral phrenic-locomotor phase relationships allowed to disclose the way the transition from alternate to synchronous phrenic coupling was achieved: synchronism appeared as resulting from a strong facilitation on the overlapping parts of the bilaterally alternating phrenic bursts; this phase shifting, vs the ipsilateral locomotor pattern, accounts for the transfer of phrenic bilateral coupling.

Relationship between phrenic and hindlimb extensor activities during fictive locomotion.

In decorticate and in spinal curarized rabbit preparations, respiratory and locomotor rhythms can be closely related (1:1 coupling between successive periods), demonstrating central relationships between the two types of pattern generators. In both preparation types, phrenic discharges are highly correlated to the extensor activities.

Influence of dietary lipids and linoleic acid amounts on fatty acid content and composition of pig serum lipoproteins.

The effects of hyperlipidic polyunsaturated fat diets (PF) and saturated fat diets (SF) versus control diet (CO) on total lipid, phospholipid (PL) and cholesteryl ester (CE) content and composition of pig serum lipoproteins were studied in Large White pigs who first underwent a PF period for 14 days followed by a CO period and third a SF feeding during a fortnight. PF and SF diets induced an increase of linoleic acid in serum total lipids, especially in HDL fraction; this increase in CE and PL involved a reverse change of oleic acid. The modifications induced by the amounts of dietary linoleic acid could be explained by an alteration of LCAT activity. The fatty acid pattern of PL which constitute the LCAT substrate lead to an enrichment of cholesterol linoleate.