Disturbed ratio of erythrocyte and plasma S-adenosylmethionine/S-adenosylhomocysteine in peripheral arterial occlusive disease.

Altered homocysteine metabolism associated with peripheral arterial occlusive disease (PAOD) may lead to impairment of vital methylation reactions through accumulation of S-adenosylhomocysteine (AdoHcy) as well as through alteration of the ratio S-adenosylmethionine (AdoMet)/AdoHcy. We determined AdoMet, AdoHcy, their ratio, and homocysteine in plasma as well as AdoMet, AdoHcy, and their ratio in erythrocytes of 61 patients with PAOD (age 49-93) and 50 healthy controls (age 41-87). Geometric mean values of plasma homocysteine, AdoMet, and AdoHcy were significantly increased in patients compared with controls (15.5 vs. 10.4 micromol/l**; 107 vs. 52.3* nmol/l; 55. 0 vs. 23.1** nmol/l, respectively; *P<0.01, **P<0.001), while the ratio of AdoMet/AdoHcy was decreased in patients (1.92 vs. 2.52*). In erythrocytes patients exhibited increased levels of AdoHcy compared with controls (309 vs. 205 nmol/l**) whereas AdoMet (3351 vs. 3732 nmol/l*) and the ratio of AdoMet/AdoHcy (11.8 vs. 19.1**) were decreased. The odds ratio (OR) for developing PAOD with decreased AdoMet/AdoHcy ratio after adjustment for kidney function was significant for erythrocyte levels < or =14.2 (OR, 7.1 (6.9-7.2, 95% CI). In addition, hematocrit levels were found to be significantly decreased in patients versus controls (0.35 vs. 0.42 l/l**) and were significantly correlated with the ratio of AdoMet/AdoHcy in erythrocytes of the patients. Since the ratio of AdoMet/AdoHcy is closely linked with the activity of numerous enzymatic methylation reactions, these results suggest that methylation may be impaired in these patients.

Platelets and cytokines in concert with endothelial activation in patients with peripheral arterial occlusive disease.

We tested the hypothesis whether circulating oncostatin-M (OSM), a cytokine that in vitro promotes fibrinogen biosynthesis and smooth muscle cell proliferation, or soluble CD40 ligand (CD40L; CD154), a leukocyte and platelet surface marker that stimulates endothelial cells, were associated: (a) with fibrinogen and other soluble cell adhesion molecules, such as P-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 and platelet-endothelial cell adhesion molecule-1; or (b) with restenosis and platelet activation in 71 patients with peripheral arterial occlusive disease undergoing peripheral angioplasty (PTA). Platelet membrane activation markers (CD62P, CD63, activated GPIIb/IIIa) were immunologically measured at 0, 1, 24 and 48 h, and 3 and 6 months after PTA. Soluble cell adhesion molecules, endothelial markers and various hemostatic variables were measured before PTA. Of the patients, 42.3% developed restenosis within 6 months, defined as a >50% reduction of the lumen at the site of balloon dilatation. Soluble CD40L was not higher in the restenosis group. Interestingly, patients with high CD40L showed significantly higher soluble VCAM-1 (P < 0.01) and thrombomodulin (P < 0.01), as well as trends for higher soluble P- and E-selectin. Platelet activation was found uniformly increased mostly at 1 and 24 h, as well as at 3 and 6 months. OSM was measurable in 53.5% (6.9 +/- 9.4 pg/ml) of the patients and undetectable in the others. No differences in the rate of restenosis was found in these two groups, which did not differ with respect to fibrinogen (3.14 +/- 1.00 versus 3.21 +/- 0.70 g/l), or the other parameters. In conclusion, soluble CD40L is associated with higher endothelial biological markers that might implicate its involvement in endothelial activation. Platelet activation, probably intermittent, might play a significant role through the expression of CD40L as a source of activation signals to the endothelial cells. Free circulating OSM does not seem to correlate directly with fibrinogen or with other acute phase reaction proteins, the synthesis of which it could influence in vitro. This might well not mean, however, that OSM lacks this activity in vivo.

Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease.

In the present study, the levels of soluble adhesion molecules P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and of other markers of endothelial activation or injury, such as thrombomodulin, von Willebrand factor (vWF), as well as homocysteine, were prospectively investigated in 71 patients (21 women, 50 men, age 68+/-13) with predominantly femoropopliteal peripheral arterial occlusive disease (PAOD, stage II-IV, Fontaine) before and after percutaneous transluminal angioplasty (PTA). Thirty patients (42.3%) developed restenosis within 6 months, defined as a > 50% reduction of the lumen diameter at the site of PTA. At entry in the study, 46% and 58% of all patients had higher than normal levels of soluble P-selectin and VCAM-1, respectively. Thrombomodulin (P < 0.01) measured at entry, was significantly higher in patients who developed late restenosis, with trends for higher values for P-selectin, VCAM-1 and vWF. The relative risks for developing restenosis were 2.41 (CI95%: 1.23-4.75) and 1.54 (CI95%: 0.98-2.72) for thrombomodulin and P-selectin, respectively. Soluble P-selectin and the severity of PAOD (Fontaine stage III/IV) were found to be statistically indicative factors for late restenosis in a logistic regression risk factor analysis with an overall predictive value of 72%. At 6 months, those who developed restenosis had also higher soluble P-selectin (P < 0.01), VCAM-1 (P < 0.05) and a trend for higher thrombomodulin. Homocysteine was elevated in 52% of the patients at entry but neither was it associated with higher restenosis rates nor did it correlate with the levels of thrombomodulin or the other adhesion molecules. These findings indicate that patients with PAOD have to a significant proportion, elevated levels of circulating soluble adhesion molecules and markers of endothelial activation occurring in concert with an ongoing atherosclerotic process.

Role of hemostatic risk factors for restenosis in peripheral arterial occlusive disease after transluminal angioplasty.

In a prospective study, the role of various hemostatic factors known to be associated with thrombotic risk was investigated in 71 patients with peripheral arterial occlusive disease (PAOD, stages II through IV, Fontaine; aged 68 +/- 13 years). Laboratory investigations were done before; 1, 24, and 48 hours after; and 3 and 6 months after percutaneous transluminal angioplasty (PTA). Thirty of 71 (42.3%) patients developed restenosis (> 50% reduction of the lumen diameter) at the site of PTA within 6 months, verified by color-coded duplex sonography. Significantly increased levels of thrombin-antithrombin III complexes (P < .01), prothrombin fragments 1 + 2 (P < .01), and D-dimers (P < .01) were found 1 hour, as well as 24 to 48 hours, after PTA. Fibrinogen (P < .01) and von Willebrand factor (P < .01) were significantly higher 48 hours after PTA. Restenotic patients as a whole had higher plasma fibrinogen (3.46 +/- 1.12 versus 2.95 +/- 0.62 g/L, P < .01) and C-reactive protein (25.4 +/- 46.7 versus 7.9 +/- 6.9 mg/L, P < .05) at baseline, as well as higher fibrinogen (P < .05) and prothrombin fragments 1 + 2 (P < .01) during months 3 to 6 after PTA. There was a nonsignificant tendency for higher values of von Willebrand factor (206 +/- 98% versus 184 +/- 100%, P = .2) at baseline in patients with restenosis, whereas tissue plasminogen activator, plasminogen activator inhibitor, coagulation screening tests, blood cell counts, and serum lipids showed no significant difference between the two groups. The relative risk for developing restenosis within 6 months while having high fibrinogen (> 2.8 g/L) or C-reactive protein at baseline was 2.80 (95% CI: 1.30-6.02, P < .01) and 1.96 (95% CI: 1.07-3.58, P < .05), respectively. Patients with critical limb ischemia (stage III/IV, Fontaine) had significantly higher fibrinogen and von Willebrand factor at repeated points of time, as well as significantly higher C-reactive protein and lower creatinine clearance at entry. In the logistic regression risk factor analysis, baseline plasma fibrinogen, C-reactive protein concentration, and the severity of the arterial disease were significantly predictive of restenosis. Our results indicate that high procoagulant factors and persistent thrombin generation of the hemostatic system might promote restenosis, particularly in patients with extended atherosclerosis. This finding suggests that new treatment strategies should be taken under consideration for patients with PAOD and PTA.

Anticardiolipin antibodies are not associated with restenosis or endothelial activation after percutaneous transluminal angioplasty.

OBJECTIVE: Restenosis following percutaneous transluminal angioplasty (PTA) continues to be a major clinical problem. Anticardiolipin antibodies (aCL) have been established as risk factors for venous or arterial thrombosis. The aim of this study was to assess: a) the influence of positive aCL upon restenosis within 6 months after PTA, b) the possibility of a seroconversion from negative to positive aCL after PTA and c) a possible link between positive aCL and endothelial activation. EXPERIMENTAL DESIGN: 71 patients (50 men and 21 women, age 68+/-13 years) with peripheral arterial occlusive disease (PAOD, Fontaine II-IV) undergoing a successful PTA entered the study and were prospectively followed for 3 and 6 months thereafter. INTERVENTIONS: PTA was carried out successfully and noninvasive grading was done with duplex scanning. Laboratory investigation included aCL, thrombin generation markers, such as thrombin-antithrombin III complexes and prothrombin fragments 1+2, as well as thrombomodulin, soluble P-selectin, E-selectin and the vascular cell adhesion molecule-1, as endothelial activation markers. RESULTS: 30/71 (42.3%) patients developed restenosis (>50% reduction of the lumen diameter) within 6 months after PTA. 9/71(12.7%), had positive aCL IgG (19-35 GPL) and/or IgM (14-103 MPL) at all three measurements. 2/9 (22.2%) of aCL positive and 28/62 (45.2%) of aCL negative patients had restenosis at 6 months after PTA (relative risk RR=0.51, 95%-Cl: 0.14-1.78, chi2 non-significant). All other parameters did not differ between aCL positive and -negative groups. CONCLUSIONS: Our findings suggest that: a) patients with PAOD have a slightly higher prevalence of positive aCL compared to the general population, but no association is evident between positive aCL and restenosis within 6 months after PTA, b) no seroconversion from negative to positive aCL occurred within 6 months after PTA, c) no association of aCL with endothelial activation markers or thrombin generation markers was found.

Discrimination between neuropeptide Y and peptide YY in the rat tail artery by the neuropeptide Y1-selective antagonist, BIBP 3226.

1. The ability of the novel, nonpeptide, neuropeptide Y (NPY) Y1-selective antagonist, BIBP 3226 ¿(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide¿, to antagonize the increase in perfusion pressure induced by NPY and peptide Y (PYY) was tested in the perfused rat tail artery, a postjunctional Y1-receptor bioassay, precontracted by 1 microM phenylephrine. 2. NPY and PYY produced a concentration-dependent enhancement of the vasoconstrictor response evoked by 1 microM phenylephrine. Although NPY and PYY are roughly equipotent, the maximal contractile response elicited by PYY was about twice that elicited by NPY. 3. Increasing concentrations of BIBP 3226 caused a parallel and rightward shift in the NPY concentration-response curve without depressing the maximal response. The contractile effect of NPY was potently inhibited in a competitive manner. The pA2 value for BIBP 3226 was 7.01 +/- 0.08, a value equivalent to that observed in the rabbit saphenous vein. Although increasing concentrations of BIBP 3226 shifted the concentration-response curve of PYY to the right without any significant decrease in the maximal vasoconstrictor response, the antagonism appeared non-competitive as the slope of the Schild plot was significantly different from unity (0.58 +/- 0.04). 4. In conclusion, these data confirm that BIBP 3226 is a potent and selective nonpeptide Y1 receptor antagonist. Moreover, they show that complex interactions occur between BIBP 3226 and postjunctional receptors activated by PYY. We postulate that BIBP 3226 might discriminate between the effects of NPY and PYY at the postjunctional level in the rat tail artery. It may be that distinct receptors for NPY and PYY exist; these may or may not allosterically interact with each other. Another working hypothesis would be that there is a single receptor complex with allosterically interacting binding sites for the two peptides.

Hyperosmolarity enhances smooth muscle contractile responses to phenylephrine and partially impairs nitric oxide production in the rat tail artery.

The respective effects of hyperosmolarity caused by impermeant solutes, such as mannitol and sucrose, on the endothelium and smooth muscles cell responses were investigated in the rat tail artery. The vessels, with or without endothelium, were infused and superfused with an isosmolar saline solution, and were repeatedly stimulated with phenylephrine. Superfusing with hyperosmolar fluid (390-420 mosm/l) produced a transient increase in the arterial basal perfusion pressure which peaked after approximately 5 min and then declined within 15 min to a stable nonsignificant value above control values in subsequent experiments. In arteries with functional endothelium, the effect of phenylephrine was about 1.9-fold larger in hyperosmotic medium compared to that in isosmotic medium. In hyperosmotic media the response was still more than twofold enhanced in endothelium-denuded vessels compared to those with endothelium. In the latter, indomethacin (10 microM) had no effect, but N omega-nitro-L-arginine methylester (L-NAME; 30 mumol/l), an inhibitor of NO production, enhanced the response to phenylephrine to reach the same magnitude of response as seen in endothelium-denuded arteries. This effect of L-NAME was antagonized by L-arginine. Relaxation induced by the NO donor SIN-1 was unchanged by hyperosmolarity, indicating that the effect of NO was not impaired. It is concluded that, in the rat tail artery, the enhancement in phenylephrine-induced contractions produced in a hyperosmolar solution is due to both an endothelium-independent increase in smooth muscle responses and a moderate decrease in the production of NO, or an NO-like factor, by the endothelium. In spite of this reduction, endothelium-derived NO still plays a major role in attenuating phenylephrine-induced contractions in hyperosmolar medium.

Effects of cyclic GMP and analogues on neurogenic transmission in the rat tail artery.

1. The effects of membrane permeable analogues of guanosine 3':5'-cyclic monophosphate (cyclic GMP), and of the NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) were investigated on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries. 2. Two 8-substituted analogues of cyclic GMP (8-bromoguanosine 3':5'-cyclic monophosphate; 8-bromo-cyclic GMP and 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate; 8-pCPT-cyclic GMP) concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. These prejunctional effects were antagonized by the cyclic AMP-dependent protein kinase (PKA) inhibitor N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5 isoquinolinesulphonamide dihydrochloride (H-89; 100 nM) but not by the cyclic GMP-dependent protein kinase (PKG) inhibitors, Rp-8-bromoguanosine 3':5'-cyclic monophosphorothioate (Rp-8-bromo-cyclic GMPS; 10 microM) or Rp-8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphorothioate (Rp-8-pCPT-cyclic GMPS; 10 microM). 3. beta-Phenyl-1,N2-ethenoguanosine 3':5'-cyclic monophosphate (PET-cyclic GMP) had no effect on stimulation-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. 4. The two 8-substituted cyclic GMP derivatives, PET-cyclic GMP and SIN-1, both decreased stimulation-induced vasoconstriction. In addition, SIN-1 relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in parallel manner to the right by Rp-8-bromo-cyclic GMPS (10 microM) and Rp-8-pCPT-cyclic GMPS (10 microM) with no change in the maximum effect, showing that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 5. It is concluded that PKA activation is involved in the noradrenaline release enhancing effect of the two 8-substituted cyclic GMP analogues, whereas a cyclic GMP/PKG-operated pathway accounts for the inhibitory effects of the cyclic GMP and its analogues on vascular smooth muscle contraction.

[Normal kidney function of the transplanted kidney in ipsilateral pelvic artery occlusion 15 years after transplantation. A case report]. / Normale Transplantatnierenfunktion bei ipsilateralem Beckenarterienverschluss 15 Jahre nach Transplantation. Ein Fallbericht.

A case of a 61-year-old women 15 years after right-sided renal transplantation is discussed. She suffered from rest pain in her right leg. Examinations revealed an occlusion of the right common iliac artery without signs of renal dysfunction or severe hypertension. After performing a extraanatomic femoro-femoral crossover bypass the patient had improved leg perfusion without a change in renal function.

Vasoconstrictor effects of various neuropeptide Y analogues on the rat tail artery in the presence of phenylephrine.

1. The increase in perfusion pressure induced by neuropeptide Y (NPY), peptide YY (PYY) and related peptides were compared in the perfused rat tail artery precontracted by a submaximal concentration (1 microM) of the vasoconstrictor, phenylephrine. 2. NPY, PYY, [Leu31,Pro34]NPY, [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) and the centrally truncated and stabilized analogues [D-Cys5,8-aminooctanoic acid7-20, Cys24]-NPY (D-Cys5-NPY) and [D-Cys7, 8-aminooctanoic acid8-17,Cys20]-NPY (D-Cys7-NPY) produced a concentration-dependent enhancement of the vasoconstrictor response induced by 1 microM phenylephrine. PYY was two times more potent than NPY and [Leu31,Pro34]NPY while ESALL-NPY, D-Cys7-NPY and D-Cys5-NPY were approximately 3, 5 and 16 times less potent than NPY respectively. NPY, D-Cys5-NPY and D-Cys7-NPY gave similar maximal responses whereas those observed for PYY, [Leu31,Pro34]NPY and ESALL-NPY were much greater than that of NPY. 3. NPY 13-36 and [des-Ser3,Lys4,Cys2,8-aminooctanoic acid3-24, D-Cys27]-NPY ([es-Ser3,Lys4]Cys2-NPY) were practically inactive at concentrations up to 3 microM, whereas [des-Ser3,Lys4,D-Cys2,8-aminooctanoic acid3-24,Cys27]-NPY ([des-Ser3,Lys4]D-Cys2-NPY), which differs from [des-Ser3,Lys4]Cys2-NPY in the disulphide bridge (a D-Cys in position 2 for [des-Ser3,Lys4]D-Cys2-NPY instead of an L-CYs for [des-Ser3,Lys4]Cys2-NPY) was a weak agonist the maximal effect of which could not be ascertained. 4. The contractile effects of [des-Ser3,Lys4]D-Cys2-NPY were additive with those of NPY and [Leu31,Pro34]NPY demonstrating that it is not a partial agonist but may simply not interact competitively with the receptor binding site for NPY. NPY and PYY interacted in a manner expected of agonists competing for the same binding site.5. PYY, NPY and [Leu31,Pro34]NPY were equipotent in displacing the I125-labelled PYY from binding sites on membranes from Y1-receptor expressing SK-N-MC cells, while the centrally truncated analogues were much less potent. The rank order of potencies for displacement of the I125-PYY binding by these peptides in SK-N-MC cells correlated with their activity in enhancing the vasoconstrictor response of phenylephrine in the tail artery. For the [des-Ser3,Lys4]D-Cys2-NPY analogue, the displacement pattern was more complex in that the displacement analysis revealed the presence of two binding sites.6. In conclusion, these data provide no evidence for other than postjunctional Y1-receptors mediating the enhancement of the contractile response elicited by phenylephrine in the perfused rat tail artery. The effects of [des-Ser3,Lys4]D-Cys2-NPY indicate that the Y1-receptor may possess an allosteric binding site.

Role of the L-arginine-NO pathway and of cyclic GMP in electrical field-induced noradrenaline release and vasoconstriction in the rat tail artery.

1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction. Dibutyryl-cyclic GMP (100 JM), a weak activator of cyclic GMP-dependent protein kinase, affected neither the pre- nor the postjunctional response to electrical field stimulation.5. These data show that an NO-like substance of endothelial origin, derived from L-arginine, attenuates vasoconstriction in the rat tail artery, whether neurally-induced or evoked by exogenous noradrenaline.Since noradrenaline release was unaltered by compounds modifying NO production, this NO-like compound acted through a postjunctional mechanism. The lack of prejunctional effects of both soluble and membrane-associated guanylate cyclase activators, despite a large effect of 8-bromo-cyclic GMP,suggests that endogenous cyclic GMP production, if present in sympathetic nerves, may not be involved in the regulation of noradrenaline release in the rat tail artery.

Depolarization of rat locus coeruleus neurons by adenosine 5'-triphosphate.

Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus. The enzymatically stable P2-purinoceptor agonist alpha,beta-methylene ATP increased the firing rate without altering the amplitude or shape of action potentials; the afterhyperpolarization following a spike was not changed either. When locus coeruleus neurons were hyperpolarized by current injection in order to prevent spontaneous firing, alpha,beta-methylene ATP produced depolarization and a slight increase in the apparent input resistance. A combined application of kynurenic acid and bicuculline methiodide failed to alter the alpha,beta-methylene ATP-induced depolarization, and tetrodotoxin only slightly depressed it. A gradual shift of the membrane potential by hyperpolarizing current injection led to a corresponding decrease, but no abolition or reversal of the alpha,beta-methylene ATP effect. In the hyperpolarized region, the current-voltage curve of alpha,beta-methylene ATP came into close approximation with, but did not cross, the control curve. Elevation of the external K+ concentration, or the intracellular application of Cs+ by diffusion from the microelectrode, depressed the response to alpha,beta-methylene ATP; external tetraethylammonium was also inhibitory. External Ba2+ and Cs+ had no effect or only slightly decreased the alpha,beta-methylene ATP-induced depolarization. A low Na+, or a low Ca2+ high Mg2+ medium, as well as the presence of Co2+ in the medium, markedly reduced or even abolished the depolarization by alpha,beta-methylene ATP. ATP itself did not produce consistent changes in the membrane potential or input resistance. However, in the presence of the P1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, ATP consistently increased the firing rate and evoked an inward current. In conclusion, P2-purinoceptor activation appears to depolarize locus coeruleus neurons by inhibiting a persistent potassium current, and at the same time opening calcium-sensitive sodium channels or calcium-sensitive non-selective cationic channels.

Excitatory effects of adenosine 5'-triphosphate on rat locus coeruleus neurones.

Pontine slices of the rat brain were used for extracellular recording of the frequency of spontaneous action potentials of locus coeruleus (LC) neurones. In the absence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP; 0.3-30 mumol/l) and 2-methylthio ATP (0.3-100 mumol/l), but not ATP (1-100 mumol/l) increased the firing rate. In the presence of DPCPX 0.1 mumol/l, all three purinoceptor agonists were active, the potency order being alpha,beta-meATP greater than 2-methylthio ATP = ATP. Preincubation of the slices with tetrodotoxin (TTX) 0.5 mumol/l decreased the spike discharge but did not alter the percent facilitatory effect of alpha,beta-meATP 30 mumol/l. There was no desensitization to alpha,beta-meATP 10 mumol/l on repeated or continuous application. Suramin 100 mumol/l selectively depressed the effect of alpha,beta-meATP 30 mumol/l without interfering with the effect of equiactive concentrations (10-100 mumol/l) of glutamic acid. The concentration-response curve of alpha,beta-meATP was shifted in a parallel manner to the right by suramin 10 mumol/l. While DPCPX 0.1 mumol/l facilitated firing, suramin 100 mumol/l did not change it. In conclusion, LC neurones may possess P2-purinoceptors of an unidentified type, which share some P2x characteristics.