Exploration of efficacy and bleeding with combined phosphoinositide 3-kinase ß inhibition and aspirin in man.

BACKGROUND: Based on animal and human data, phosphoinositide 3-kinase (PI3K)ß is a promising antithrombotic target. However, the relation between efficacy and bleeding when combined with current antiplatelet therapies is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the short-acting inhibitor AZD6482 alone and in different combinations with P2Y12 and cyclooxygenase (COX)-1 inhibition in vitro (human platelets), in vivo (dog), and in healthy subjects. METHODS AND RESULTS: Evaluation of complete target inhibition of PI3Kß (by AZD6482), P2Y12 (by ticagrelor), and COX-1 (by aspirin) alone and in the different combinations vs. concentration responses for a panel of platelet agonists in vitro (adenosine diphosphate, collagen, thrombin receptor activating peptide) indicates that the rank order of antiplatelet efficacy is P2Y12  > PI3Kß > COX-1 as monotherapy and P2Y12 plus PI3Kß > P2Y12 plus COX-1 > PI3Kß plus COX-1 as dual therapy, with little additional effect with triple therapy. Use of a conscious dog model to assess ex vivo antiplatelet effect in parallel with bleeding time prolongation (standard incision in the ear) confirms the wide separation of efficacy vs. bleeding for PI3Kß inhibition and that this separation is reduced when combined with aspirin and more reduced when combined with clopidogrel. In healthy subjects, AZD6482, in combination with aspirin, shows a potential for greater antiplatelet potency but less bleeding potential compared with clopidogrel plus aspirin. CONCLUSIONS: PI3Kß inhibition, in comparison with P2Y12 and COX-1, delivers medium antiplatelet effect but with minimal bleeding. PI3Kß inhibition, in combination with aspirin, in healthy subjects, provides a potential for greater overall antiplatelet effect compared with clopidogrel plus aspirin, but with significantly less bleeding potential.

Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B.

BACKGROUND: The present study was carried out to investigate the impact of FII levels, and their increase, on the hemostatic potential in plasma from hemophilia A and B patients with and without inhibitors. METHOD: Recombinant human factor (F) II (rhFII) was added ex vivo to plasma from 68 patients with hemophilia A and B, with or without inhibitors. The hemostatic potential as measured by thrombin generation (calibrated automated thrombogram [CAT]) was focused on the endogenous thrombin potential (ETP) as it has been shown to correlate with the clinical phenotype of bleeding in hemophilia patients and has also been used to guide bypassing therapy in hemophilia patients with inhibitors before elective surgery. The factor eight inhibitor bypassing agent (FEIBA(®) ) was used as a reference to the clinical situation. RESULTS: The study shows that rhFII concentration-dependently increased ETP by a similar magnitude in hemophilia A and B, both with and without inhibitors. Compared with FEIBA, rhFII showed a shallower concentration-response curve. In both types of hemophilia 100 mg L(-1) of rhFII roughly doubled the ETP. A corresponding response was obtained by 0.5 U mL(-1) of FEIBA. CONCLUSION: These data support the theory that FII is one of the major components responsible for the efficacy of FEIBA. The data also indicate that rhFII may be useful, alone or in combination with other coagulation factors, in some of the conditions for which FEIBA is used today, although more data are needed to substantiate this.

Human target validation of phosphoinositide 3-kinase (PI3K)ß: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kß inhibitor.

BACKGROUND: Based on in vitro and animal data, PI3Kß is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the novel, short-acting inhibitor AZD6482. METHODS AND RESULTS: AZD6482 is a potent, selective and ATP competitive PI3Kß inhibitor (IC(50) 0.01 µm). A maximal anti-platelet effect was achieved at 1 µm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti-thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3-h infusion. The ex vivo anti-platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin-induced human adipocyte glucose uptake in vitro (IC(50) of 4.4 µm). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 µm but reduced by about 60% at a plasma exposure of 27 µm. In man, the homeostasis model analysis (HOMA) index increased by about 10-20% at the highest plasma concentration of 5.3 µm. CONCLUSIONS: This is the first human target validation for PI3Kß inhibition as anti-platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at 'supratherapeutic' plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.

High leucovorin doses during high-dose methotrexate treatment may reduce the cure rate in childhood acute lymphoblastic leukemia.

We explored the relationship between time to relapse and different exposure variables (serum methotrexate (S-MTX) 23, 36 and 42 h after start of administration, MTX elimination time and leucovorin (LV) dose) during high-dose MTX (HDM) treatment of 445 children with acute lymphoblastic leukemia. MTX was infused at 5 g/m2 (non-high risk) or 8 g/m2 (high risk) over 24 h, 2-9 times per patient. LV rescue dose was adjusted according to the S-MTX concentration. Time from end of the last HDM to relapse was analyzed by Cox regression analysis with the logarithms of S-MTX and LV dose as exposures. The combined results from all risk groups suggest that high LV dose is related to higher risk for relapse. Doubling of the LV dose increased the relapse risk by 22% (95% confidence interval 1-49%, P = 0.037). High LV doses correlated with high MTX levels at 23, 36 and 42 h and longer elimination time. The results suggest that high doses of LV increase the risk for relapse despite the fact that they were correlated with high MTX levels and longer MTX elimination time. The choice of MTX and LV doses may be regarded as an intricate balance between effect and counter-effect.

High-dose methotrexate: on the relationship of methotrexate elimination time vs renal function and serum methotrexate levels in 1164 courses in 264 Swedish children with acute lymphoblastic leukaemia (ALL).

PURPOSE: The objectives of the present study were to determine the relationship between methotrexate (MTX) elimination time and various aspects of renal function and to evaluate the prognostic value of elevated serum MTX and creatinine for delayed MTX elimination. PATIENTS AND METHODS: The majority of the 264 children were being treated for ALL. According to the NOPHO-92 protocol, 5 or 8 g MTX/m(2) was administered over 24 h. Serum creatinine was assessed daily. In 11 patients from one centre, renal function was studied in more detail using serum cystatin C, iohexol clearance, and urinary albumin, IgG and protein HC. RESULTS: Increased serum creatinine correlated significantly with the elimination time of MTX, whereas no indications were found of tubular or barrier function damage. Of the 1164 courses, 44 had delayed elimination of MTX (>/=120 h). Serum MTX >150 microM at the end of infusion had a sensitivity of 0.27 and a specificity of 0.94 to predict delayed MTX elimination, and >/=50% increase in serum creatinine during the first treatment day (creatinine ratio) had a sensitivity of 0.32 and a specificity of 0.99. The corresponding risk ratios were 5 and 19 for MTX >150 micro M and creatinine ratio, respectively. In courses with a normal elimination time (<72 h), 99% of the courses had a rise in serum creatinine of less than 50%. CONCLUSIONS: Elevation of serum creatinine by more than 50% is a better predictor of delayed elimination than the level of serum MTX at the end of MTX infusion, especially if information on previous creatinine measurements is used to reduce the impact of an occasionally low serum creatinine value before the start of the MTX infusion.

Sample handling for determination of free platinum in blood after cisplatin exposure.

Cisplatin is a commonly used cytostatic drug that can be pharmacokinetically monitored by measurement of non-protein-bound platinum (Pt) in plasma. The present report elucidates some practical aspects on blood sample preparation with the purpose of identifying methodological error sources and simplifying sample handling. Human blood was incubated in vitro with clinically relevant doses of cisplatin and then stored at different temperatures for various periods prior to deproteinization. Pt concentrations were analyzed by inductively coupled plasma mass spectrometry, which is a very sensitive technique that is well suited for determination of free Pt in biological samples. Free Pt concentration is usually determined after ultrafiltration of plasma. An alternative approach used to study the protein-free fraction is to precipitate the proteins with ice-cold ethanol and then analyze the aliquot. We compared the Pt concentrations in protein-free plasma obtained by these two methods and the levels were very similar. Thus, ethanol deproteinization may be an attractive alternative to ultrafiltration, since it is both simple to perform and very cost-effective. Storage of nondeproteinized whole blood or plasma at 4 degrees C or 20 degrees C did not affect the free Pt fraction significantly for the first 2 h, after which a gradual decrease was noted. This suggests that saving the blood sample for approximately 1 h before processing should not have any major impact on the result. On long-term storage of frozen blood samples prior to deproteinization the freezing temperature proved to be of critical importance. At -20 degrees C there was a gradual decrease in the free Pt fraction during the study period of 14 days, whereas the free Pt concentration remained unchanged at -70 degrees C for at least 3 months. Thus, samples may be stored unprocessed at -70 degrees C for several months, whereas -20 degrees C is not sufficiently cold.

Differential effects of calcium antagonists and Bay K 8644 on contractile responses to exogenous noradrenaline and adrenergic nerve stimulation in the rabbit ear artery.

1. The effects of three calcium antagonists (nifedipine, verapamil, diltiazem) and the calcium agonist Bay K 8644 were compared on contractile responses of similar amplitude elicited by noradrenaline (NA) and electrical nerve stimulation (ENS) in the rabbit isolated ear artery. 2. Contractions induced by both NA (3 x 10(-7) M) and ENS (10 Hz, 10s) were almost exclusively mediated by alpha 1-adrenoceptors, since 10(-7) M prazosin abolished (NA) or almost abolished (ENS) the responses, and prazosin was more than three orders of magnitude more potent than rauwolscine on both types of response. 3. ENS-induced contractions were considerably less inhibited by nifedipine, verapamil and diltiazem than were those elicited by NA. Bay K 8644 enhanced responses to NA more than those to ENS. 4. The inhibitory effect of nifedipine and Ca2+ deprivation on NA-induced contractions decreased with increasing NA concentration. Reduction of the NA response by prazosin or phenoxybenzamine increased the nifedipine inhibition. 5. Reduction of the ENS-induced contractions by prazosin or phenoxybenzamine, or by use of a lower stimulation frequency did not increase the inhibitory effect of nifedipine. 6. In conclusion, the differential effects of the calcium antagonists on NA- and ENS-induced contractions were not related to differences in alpha-adrenoceptor subtype (alpha 1/alpha 2), receptor reserve or response amplitude, but may rather reflect temporal and spatial differences in alpha-adrenoceptor activation between the responses.

Influence of Bay K 8644 on vascular responses mediated by alpha 1- and alpha 2-adrenoceptors.

1. The calcium channel activator Bay K 8644 increased the potency of noradrenaline in cat middle cerebral (alpha 2-adrenoceptors) and mesenteric (atypical or mixed alpha 1- and alpha 2-adrenoceptor population) arteries, but not in rat middle cerebral and mesenteric arteries (alpha 1-adrenoceptors). 2. In cat arteries, exposure to 15 mM K+ solution shifted the noradrenaline concentration-response curve to the left in an almost identical manner as did Bay K 8644. 3. Bay K 8644 completely reversed the relaxation produced by nifedipine in K+-contracted cat middle cerebral arteries, whereas the relaxation induced by verapamil, diltiazem or flunarizine was only partially reversed. This suggests a specific interaction between Bay K 8644 and the dihydropyridine receptors on the calcium channels. 4. It is concluded that the degree to which noradrenaline promotes calcium influx through membrane channels is at least partly related to the alpha-adrenoceptor subtype mediating the response.

Theoretical and functional studies on alpha 1-and alpha 2-adrenoreceptors: an examination using the Schild plot.

1 The influence on the shape of the Schild plot by a two-receptor system was studied in both functional and theoretical studies. In the functional studies, the alpha-adrenoreceptors in cat lingual arteries were studied since both alpha 1- and alpha 2-adrenoreceptors have been suggested to contribute to the noradrenaline-induced contractile response in this tissue. 2 The Schild plots constructed using noradrenaline (NA) as agonist and prazosin as antagonist gave a straight line with a slope close to unity. In contrast, the corresponding Schild plot constructed for rauwolscine appeared to be biphasic. 3 The results obtained in the functional study were discussed in view of Schild plots obtained from a theoretical model which was designed to take into consideration the presence of two distinct types of receptor, the percentile proportions of which could be altered. 4 The theoretical model indicates that the shape of the Schild plot can vary considerably depending on the relative contribution of each receptor subtype to the contractile response, the selectivity of the antagonist, and the range and number of antagonist concentrations used. 5 It is suggested that a response is predominantly mediated by alpha 1-adrenoreceptors and less by alpha 2-adrenoreceptors when the Schild plot for prazosin gives a slope close to unity and has a pA2-value representative for alpha 1-adrenoreceptors, and rauwolscine gives a slope less than unity and a significant shift of the NA concentration-response curve in concentrations around 10(-8) M. When the reverse is true (but with a significant shift caused by prazosin already at a concentration around 10(-9) M) the contraction elicited by NA is proposed to be predominantly mediated by alpha 2-adrenoreceptors and less by alpha 1-adrenoreceptors.

Postjunctional alpha-adrenoceptors in human superficial epigastric arteries and veins.

A pharmacological characterization of the postjunctional alpha-adrenoceptors in human superficial epigastric artery and vein was performed, using several alpha-adrenoceptor subtype selective agonists, and the antagonists prazosin (alpha 1) and rauwolscine (alpha 2). In the arteries prazosin fulfilled the criteria for a competitive antagonism in concentrations 10(-9)-10(-7) M, giving a pA2-value of 9.17 in the Schild plot. Rauwolscine in concentrations 10(-8)-10(-6) M caused less pronounced but significant dextral shifts of the noradrenaline (NA) concentration-response curves. In the veins rauwolscine behaved like a competitive antagonist (10(-8)-10(-7) M). The pA2-value was 9.16. Prazosin 10(-9) M displaced the NA concentration-response curve, but higher concentrations (10(-8) and 10(-7) M) caused no further displacement. Prazosin reduced the Emax-values in the veins. In the arteries the rank order of potency for the agonists was: cirazoline (alpha 1) greater than NA greater than naphazoline (alpha 2) greater than guanfacine (alpha 2) greater than phenylephrine (alpha 1). The intrinsic activities of clonidine (alpha 2), ST 587 (alpha 1), B-HT 920 (alpha 2) and B-HT 933 (alpha 2) were too low to allow meaningful comparisons to be made. The rank order of potency in the veins was: NA greater than clonidine (alpha 2) greater than naphazoline (alpha 2) greater than guanfacine (alpha 2) greater than phenylephrine (alpha 1) greater than B-HT 920 (alpha 2) greater than cirazoline (alpha 1) greater than B-HT 933 (alpha 2). The intrinsic activity of ST 587 was low.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alpha-adrenoceptor subtype-selective antagonists on the human saphenous vein in vivo.

The effects of the alpha-adrenoceptor subtype-selective antagonists prazosin (alpha 1) and yohimbine (alpha 2) on the saphenous vein of six healthy male subjects were investigated in vivo. The drugs were infused locally into the congested (40 mmHg), long saphenous vein constricted by simultaneous local infusion of noradrenaline (NA). Prazosin 10(-9) M (concentration in the infusion solution, infusion rate 0.3 ml min-1) did not reduce the NA-induced venoconstriction, but at a concentration of 10(-8) M there was a significant reduction; in two subjects no response to NA could be elicited in the presence of 10(-8) M prazosin. Prazosin 10(-7) M caused no further reduction of the NA effect compared to that produced by 10(-8) M in three of the subjects, whereas in one, prazosin 10(-9), 10(-8) and 10(-7) M caused a dose-dependent blockade. Yohimbine, 10(-9), 10(-8) and 10(-7) M caused a dose-dependent reduction of the NA-induced venoconstriction in all subjects. The results suggest that the human saphenous vein is endowed with functionally important populations of both alpha 2- and alpha 1-adrenoceptors.

Influence of extracellular calcium and nifedipine on alpha 1- and alpha 2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries.

The influence of extracellular Ca2+ and nifedipine on contractile responses to 10 microM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly alpha 2-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly alpha 1-adrenoceptors) and CMA (sensitive to both alpha 1- and alpha 2-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca2+-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K+ was more sensitive to Ca2+ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca2+, can also release Ca2+ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, alpha 1-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca2+ influx and intracellular Ca2+ release, whereas alpha 2-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca2+ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca2+-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca2+, whereas in the other types of artery, Ca2+ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca2+ influx and intracellular Ca2+ release to the contractile activation, but also in the nifedipine sensitivity of the Ca2+ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of alpha-adrenoceptor stimulated by NA and to the type of vessel studied.

Cerebrovascular responses to haemorrhagic hypotension in anaesthetized cats. Effects of alpha-adrenoceptor antagonists.

Haemorrhagic hypotension induces the phenomenon of cerebrovascular autoregulation and, concomitantly, involves an activation of the sympathetic nervous system. As brain vessels in cats have an atypical adrenoceptor distribution we studied the effects of an alpha-adrenoceptor antagonist on the autoregulatory response to haemorrhage. Cortical blood flow was studied by the H2 technique in chloralose-anaesthetized cats subjected to a period of graded haemorrhage over 3 h. Three groups of cats were studied: control, i.e. those receiving saline (n = 10); yohimbine-treated (200 micrograms . kg-1 . h-1, n = 7); and prazosin-treated (50 micrograms . kg-1 . h-1, n = 6). In the control group, cortical blood flow remained relatively constant when mean arterial pressure was decreased from 102 +/- 1 mmHg (mean +/- SE) to approximately 50 +/- 1 mmHg; thereafter, blood flow decreased with decreasing perfusion pressure. In the arterial pressure range 64-55 mmHg, cortical blood flow was significantly higher in the yohimbine group (109 +/- 12 ml . 100 g-1 . min-1) compared to the control group (69 +/- 6 ml . min-1) and remained higher in the yohimbine-treated cats at more extreme levels of hypotension. Blood flow did not fall significantly in the yohimbine-treated cats until mean arterial pressures of 31 +/- 1 mmHg were attained. In the prazosin-treated cats, flow began to decrease at arterial pressures even greater than those observed in the control group. Thus, there is a sympathetic vasoconstriction of brain arteries that is primarily mediated by alpha 2-adrenoceptors in the feline cerebrovascular bed.

Contraction-mediating alpha-adrenoreceptors in isolated human omental, temporal and pial arteries.

The alpha-adrenoreceptors mediating contraction in human omental (OA), temporal (TA), and pial (PA) arteries obtained during surgery, were characterized by means of subtype selective agonists and antagonists. In the OAs and TAs, prazosin concentration-dependently shifted the noradrenaline (NA) concentration-response (cr) curve towards higher concentrations, without depression of maximum. The corresponding Schild plots had slopes close to unity. Also rauwolscine caused a rightward displacement of the NA cr-curve in both OAs and TAs, without affecting the maximum response. In the TAs, OAs, rauwolscine 3 x 10(-8) M shifted the curve and the Schild plot seemed to be biphasic. Oxymetazoline, but not clonidine, produced contractile responses in the TAs and OAs, and phenylephrine was a full agonist in both types of vessel. The PAs showed a pronounced inter- and intra-individual variation in the response to NA, and often exhibited spontaneous activity. Prazosin was considerably more effective than rauwolscine and yohimbine to inhibit NA-induced responses. Clonidine had no contractant effect, whereas, oxymetazoline was more, and phenylephrine less potent than NA. It is concluded that in human OAs, TAs and PAs, the alpha-adrenoreceptor mediating contraction is mainly of the alpha 1-type.

Postjunctional alpha 1- and alpha 2-adrenoceptors mediating contraction in isolated human groin arteries and veins.

By means of selective agonists and antagonists for alpha 1- and alpha 2-receptors, the alpha-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the alpha 1-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA2 = 9.86); the selective alpha 2-receptor antagonist rauwolscine in the concentration 10(-8) M caused a right-ward shift of the NA cr-curve without reduction of Emax, but 10(-7) M and 10(-6) M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA2 = 9.03); prazosin 10(-9) M significantly displaced the NA cr-curve, whereas 10(-8) M and 10(-7) M caused little or no further shift. The responses to the alpha 2-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC50 = 6.24). Phenylephrine, selective for alpha 1-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of alpha 1-receptors in the arteries and of alpha 2-receptors in the veins.

The postjunctional alpha-adrenoceptors of the human saphenous vein.

The postjunctional alpha-adrenoceptors in human long saphenous vein were characterized using alpha-adrenoceptor subtype selective agonists and antagonists. The order of potency for the alpha-adrenoceptor agonists used was: Clonidine (alpha 2) greater than BHT-920 (alpha 2) greater than naphazoline (alpha 2) greater than guanfacine (alpha 2) greater than cirazoline (alpha 1) greater than phenylephrine (alpha 1) greater than ST 587 (alpha 1) greater than BHT-933 (alpha 2). Clonidine had the same potency as noradrenaline (NA), but was 52 times more potent than phenylephrine (pEC50 7.09 and 5.37, respectively). Phenylephrine and guanfacine had intrinsic activities that did not differ from that of NA, whereas the intrinsic activities of the other agonists were significantly lower. The highly selective alpha 1-adrenoceptor antagonist prazosin in concentrations 10(-9)-10(-7) M was unable to cause a significant shift of the NA concentration response (cr) curve to the right. However, the alpha 2-adrenoceptor antagonists yohimbine and rauwolscine in concentrations 10(-8)-10(-6) M shifted the NA cr-curve towards higher concentrations. No concentration of any antagonist used significantly attenuated the maximum contraction. The slope of the regression line in the Schild plot differed significantly from unity for rauwolscine but not for yohimbine, and the pA2-values were 9.00 and 8.27, respectively. These results suggest that the contraction mediating alpha-adrenoceptors in the human saphenous vein are mainly of the alpha 2-type. However, the low slope value of the Schild plot for rauwolscine may indicate the presence of a small population of alpha 1-adrenoceptors.

Pharmacological properties of prejunctional alpha-adrenoceptors in isolated feline middle cerebral arteries; comparison with the postjunctional alpha-adrenoceptors.

In cat middle cerebral arteries (CMCA) preincubated with 3H-noradrenaline (NA), the outflow of tritium evoked by electrical stimulation was reduced to 32% by alpha-adrenoceptor (alpha-receptor) stimulation with oxymetazoline, and increased to 487% by alpha-receptor blockade with HEAT. The relative order of potency for alpha-receptor agonists on prejunctional receptors was: clonidine greater than or equal to oxymetazoline greater than phenylephrine, and the antagonist rauwolscine was more potent than prazosin. This indicates that the prejunctional alpha-receptors are mainly of alpha 2-type. Rauwolscine was more potent than prazosin in inhibiting the contractions induced by NA, indicating a predominance of alpha 2-receptors postjunctionally. Apart from clonidine having higher intrinsic activity pre- than postjunctionally, all drugs examined (oxymetazoline, phenylephrine, rauwolscine, HEAT (BE2254), and prazosin) had similar concentration-effect curves on the pre- and postjunctional receptors. Furthermore, the ratios of EC50-values pre- and postjunctionally of rauwolscine, oxymetazoline, and clonidine were all close to unity. These results indicate that pre- and postjunctional alpha 2-receptors in the CMCA have similar pharmacological characteristics and cannot be influenced separately by the presently used drugs.

Interaction of peptides present in perivascular nerves on the smooth muscle of cat cerebral arteries and on the stimulation-evoked efflux of 3H-noradrenaline.

Studies have been performed using isolated segments of the middle cerebral artery of the cat to determine whether noradrenaline (NA), neuropeptide Y (NPY), avian pancreatic polypeptide (APP), substance P (SP), vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) have effects that involve pre- and/or postsynaptic mechanisms. NA, clonidine, NPY and APP produced concentration-dependent contractions of cerebral artery segments, whereas SP and VIP induced relaxation of prostaglandin F2 alpha-contracted arteries. GRP, in the concentrations tested, (up to 2 X 10(-7)M) had no vasomotor activity. The alpha 2-adrenoreceptor agonist clonidine (10(-8) and 10(-6)M) produced a reduction in the field stimulation-evoked release of 3H-NA previously incorporated into the noradrenergic transmitter pool of the arteries, whereas the alpha 2-adrenoreceptor antagonist rauwolscine (10(-8) and 10(-6)M) resulted in increases in 3H-NA release. However, none of the peptides caused any significant change of the stimulation-evoked fractional release of 3H-NA. The results suggest that NPY, APP, SP and VIP have postjunctional effects in cat cerebral arteries and, in the concentrations tested do not affect stimulation-evoked NA release.

Pharmacological characterization of postjunctional alpha-adrenoceptors in isolated human omental arteries and veins.

The alpha-adrenoceptors in human omental arteries and veins were characterized and compared. In the arteries both prazosin (pA2 9.48) and rauwolscine (pA2 7.19) displaced the noradrenaline (NA) concentration-response (cr) curve towards higher concentrations without reduction of maximum. Neither clonidine, nor oxymetazoline had any consistent contractile effects. Phenylephrine had a lower potency than NA, but a similar intrinsic activity. In the veins, both prazosin (pA2 9.72) and rauwolscine (pA2 8.11) displaced the NA cr-curve towards higher concentrations, but also significantly depressed maximum. Clonidine and oxymetazoline contracted veins from 3 out of 7 and 4 out of 6 patients, respectively. Their pD2-values were similar to that of NA, but their intrinsic activities were significantly lower. NA was more potent than phenylephrine in these vessels, and there was no significant difference in intrinsic activity. The results suggest that in human omental arteries, the postjunctional alpha-adrenoceptors are mainly of the alpha 1-type, even if a small population of alpha 2-adrenoceptors cannot be excluded. In omental veins, there seems to be a functionally important population of postjunctional alpha 2-adrenoceptors occurring together with a population of alpha 1-adrenoceptors.

Presence of alpha-adrenoceptors in human temporal arteries. Comparison between migraine patients and controls.

Alpha-adrenergic mechanisms have frequently been implied in migraine pathophysiology. We have examined the noradrenaline reactivity of isolated human temporal arteries removed from six migraine sufferers (not during attack) and from six patients without migraine operated for intracranial disorders. Noradrenaline constricted these vessels in a concentration-dependent manner, the response being altered by phentolamine 10(-8) M to 10(-6) M. There was no statistically significant difference between migraine patients and controls with respect to maximal contractile force (Emax) or pD2 (negative logarithm of the concentration eliciting half maximal force). The pA2 value for phentolamine was 8.3 in vessels from controls and 7.6 in arteries from migraine sufferers. The small difference between migraine patients and controls was not statistically significant. We obtained clear evidence of alpha-adrenergic receptors in human temporal arteries but their sensitivity was independent of the migraine disorder.