(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets.

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .

Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland.

1. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to increase cyclic AMP production in dispersed cell aggregates from the major salivary glands of the rat. The goal of the present study was to identify the 5-HT receptor subtypes that mediate these effects in rat submandibular glands (SMG). 2. Among the 5-HT receptor subtypes identified in the rat, 5-HT(4(a,b)), 5-HT(6) and 5-HT(7(a,b,c)) activate adenylyl cyclase (AC). We used subtype specific primers to screen rat SMG by reverse transcription-PCR. Results indicate the presence of mRNA for 5-HT(4(b)) and 5-HT(7(a)) but not for 5-HT(4(a)), 5-HT(6) and 5-HT(7(b,c)). 3. In dispersed SMG cells, 5-carboxyamidotryptamine (5-CT), a 5-HT(7) receptor agonist, stimulated cyclic AMP synthesis with higher potency (EC(50)=27+/-5 nM) but lower efficacy than 5-HT, suggesting a 5-HT(7) component and an additional component in the response to 5-HT. The 5-HT(7) contribution was further supported by antagonism of the 5-CT effect by metergoline, a 5-HT(7) antagonist, which exhibited an affinity (K(i)=50 nM) similar to that obtained at the cloned 5-HT(7) receptor. 4. In the presence of a maximally effective concentration of 5-CT, 5-HT produced an additional increase in cyclic AMP production that was inhibited by the 5-HT(4) receptor antagonist, GR113808, suggesting that the second component of cyclic AMP production is mediated by 5-HT(4) receptors. 5. These findings indicate the presence in rat SMG of both 5-HT(4(b)) and 5-HT(7(a)) receptors positively coupled to AC.

Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.

1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostaglandin E2(PGE2) in the paw exudate. 3 Paw oedema was maximal by 6 h and remained elevated for 10 h following carrageenan administration. The non-selective cNOS/iNOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) given intravenously (30-300 mg kg-1) 1 h before or after carrageenan administration, inhibited paw oedema at all time points. 4 The selective iNOS inhibitors, N-iminoethyl-L-lysine (L-NIL) or aminoguanidine (AG), failed to inhibit carrageenan-induced paw oedema during the first 4 h following carrageenan administration, but inhibited paw oedema at subsequent time points (from 5-10 h). iNOS mRNA was detected between 3 to 10 h following carrageenan administration using ribonuclease protection assays. iNOS protein was first detected 6 h and was maximal 10 h following carrageenan administration as shown by Western blot analysis. Administration of the iNOS inhibitors 5 h after carrageenan (a time point where iNOS was expressed) inhibited paw oedema at all subsequent time points. Infiltrating neutrophils were not the source of iNOS since pretreatment with colchicine (2 mg kg-1) suppressed neutrophil infiltration, but did not inhibit the iNOS mRNA expression or the elevated NO2-/NO3- levels in the paw exudate. 5 Inhibition of paw oedema by the NOS inhibitors was associated with attenuation of both the NO2-/NO3- and PGE2 levels in the paw exudate. These inhibitors also reduced the neutrophil infiltration at the site of inflammation. 6 Recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (PEGrhSOD; 12 x 10(3) u kg-1), administered intravenously either 30 min prior to or 1 h after carrageenan injection, inhibited paw oedema and neutrophil infiltration, but had no effect on NO2-/NO3- or PGE2 production in the paw exudate. The administration of catalase (40 x 10(3) u kg-1), given intraperitoneally 30 min before carrageenan administration, had no effect on paw oedema. Treatment with desferrioxamine (300 mg kg-1), given subcutaneously 1 h before carrageenan, inhibited paw oedema during the first 2 h after carrageenan administration, but not at later times. 7 These results suggest that the NO produced by cNOS is involved in the development of inflammation at early time points following carrageenan administration and that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time points. The potential interactions of NO with superoxide anion and PG is discussed.

Evidence of peroxynitrite involvement in the carrageenan-induced rat paw edema.

The role of peroxynitrite generated from nitric oxide and superoxide anion was investigated in a model of acute inflammation induced by the injection of carrageenan into the rat hind paw. Paw edema was inhibited 8 h following the administration of carrageenan by N-iminoethyl-L-lysine (3-30 mg/kg, n = 6) or aminoguanidine (30-300 mg/kg, n = 6), two selective inhibitors of inducible nitric oxide synthase and by recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (12 x 10(3) U/kg, n = 6, P < 0.001). Moreover, at the same time point following carrageenan administration, intense immunoreactive staining for nitrotyrosine (a marker of peroxynitrite formation) was detected. Our results suggest that the generation of nitric oxide, superoxide anion and peroxynitrite contributes to the edema observed in this acute model of inflammation.

[Evaluation of pulmonary arterial hypertension by Doppler echocardiography in chronic respiratory insufficiency]. / Evaluation de l'hypertension artérielle pulmonaire par échocardiographie doppler dans l'insuffisance respiratoire chronique.

The usefulness of doppler-echocardiography for the assessment of pulmonary arterial hypertension in patients with chronic respiratory failure was evaluated in 24 consecutive patients with chronic obstructive lung disease. Seventeen of these 24 patients (71 p. 100) who had tricuspid valve regurgitation analysable by the continuous wave doppler technique were selected as study group; they included 15 men and 2 women aged from 33 to 78 years (mean 63 years). The highest maximum velocity value (method A) or the maximum velocity value averaged on several cycles (method B) of the tricuspid regurgitation jet was used to calculate the right ventriculo-atrial pressure gradient, using Bernouilli's equation. Right atrial pressure was determined by three methods: haemodynamic measurement, clinical evaluation or attribution of an arbitrary 10 mmHg value. The pulsed doppler study of the pulmonary ejection flow included measurement of the acceleration time and calculation of the acceleration time/ejection time ratio. The usual echocardiographic parameters were measured. Catheterization was performed 2.5 days on average after the doppler study. Correlations between doppler examination and catheterization to evaluate the right ventricular systolic pressure were significant (p less than 0.001) and better with method B than with method A. Depending on the method employed to evaluate the right atrial pressure, the correlation coefficients obtained with method B were: 0.93 (haemodynamic measurement), 0.91 (clinical evaluation) and 0.88 (arbitrary value of 10 mmHg). The right ventricular systolic pressure evaluated by doppler ultrasound using method B and by clinical evaluation of the right atrial pressure was 47 +/- 12 mmHg (22 to 70 mmHg), as against 51 +/- 13 mmHg (28 to 74 mmHg) measured by catheterization.(ABSTRACT TRUNCATED AT 250 WORDS)

[Enzymatic activities in the liver, and muscle nucleic acids, in swine receiving an unbalanced diet with an excess of methionine for 100 days]. / Nutrition animale . -Activités enzymatiques dans le foie, at acides nucléiques musculaires, chez des porcs recevant pendant 100 jours une alimentation déséquilibrée par excès de méthionine.

Twelve growing Swine were fed an 18% protein diet (Maize and Soja bean) for one hundred days containing either 0,6% sulfur amino acids (basal diet) or 0,6% and 1% DL-methionine added to the control diet. Such an excess, reduced food intake and body weight gain mainly during the "finishing period" (60 to 100 kg). The RNA/DNA and protein/DNA ratios in the muscle did not show any difference. Hepatic activities of some enzymes involved in glycolysis, gluconeogenesis and amino acid metabolism, were unchanged, except that of methionine adenosyl transferase, the first step of transsulfuration, which was induced in proportion with the amount of the methionine ingested. Swine seemed to adapt to the excessive methionine intake, which did not show any toxicity in our experimental conditions.

Exclusion of pancreatic exocrine secretion from intestine in the pig: existence of a digestive compensation.

The apparent digestibility of a diet was studied over a period of 87 days after ligature of the pancreatic duct, in the growing pig. Sham operated pigs were used as controls. Feces and urine collections were made during three periods of 10 days each. Mean daily gain of sham operated pigs was higher (560 g/day) than that of pancreatic duct ligated pigs (421 g/day), throughout the experiment. Within the first period (15-24 days after ligature), the apparent digestibility of nitrogen was most affected (-35.6%) while that of energy decreased by 12.1% as compared to values obtained in sham operated pigs. Nitrogen retention was similar in all pigs. Between the 1st (15-25 days) and the 3rd period (78-87 days) after exclusion of the pancreatic secretion from intestine, the apparent digestibility of nitrogen increased by 20% and that of energy by 6%, which might suggest digestive compensation. At the end of the experiment, weights of empty small intestine and liver were significantly higher in the pancreatic duct ligated pigs. The origin of the digestive compensation is discussed.