Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Summary.  The integrin αvβ3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates αvβ3 activity in platelets, we measured the effect of the P2Y₁ antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y₁₂ antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y₁ and P2Y₁₂ was required to activate αvβ3. The reducing agent dithiothreitol and Mn²⁺ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates αvβ3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, αvβ3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Gα_q- and Gα_i-coupled receptors converge to release cytoskeletal constraints on αvβ3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for αvβ3-mediated platelet adhesion.     

β-adrenergic priming of rats in vivo modulates the effect of β-agonist in vitro on surfactant phospholipid metabolism of isolated lungs

Abstract. To evaluate the effects of multiple β -adrenergic stimulations on pulmonary surfactant phospholipids, perfused lungs from β -adrenergic primed and non-primed rats were challenged with the β -agonist terbutaline in vitro . Cell-free lung lavage, lavagable alveolar cells and lung tissue were analysed for phospholipid content and incorporation of precursors. In lung lavage, terbutaline in vitro doubled the incorporation of ¹⁴C-choline and ³H-palmitate into total phosphatidylcholine (PC) and of ³H-palmitate into phosphatidylglycerol (PG). β -adrenergic priming in vivo prior to terbutaline in vitro lowered the increase of precursor incorporation. For lavagable cells, terbutaline in vitro increased the incorporation of ³H-palmitate into PC. Priming in vivo reduced this effect and diminished the specific ³H-choline incorporation into lavagable cell PC below control level. For lung tissue, priming increased the amounts of PC and disaturated PC (DSPC) whereas terbutaline in vitro decreased DSPC in both primed and non-primed lungs. Terbutaline in vitro slightly increased the incorporation of ¹⁴C-choline and ³H-palmitate into PC and DSPC in non-primed but not in primed lungs. β -adrenergic blockade by ICI 118.551 prevented all effects but generally increased ³H-palmitate incorporation into the phospholipids and, in lavagable cells, the amount of PC. We conclude that long-term β -adrenergic treatment may alter the metabolism of pulmonary surfactant phospholipids by increasing tissue PC and DSPC and by decreasing the secretion of newly-synthesized PC.     

Reduced β-adrenergic sensitivity in healthy volunteers induced by hypoglycemia

Summary— A single causative mechanism for development of hypoglycemia unawareness in insulin-dependent diabetes mellitus (IDDM) is not yet apparent. Reduced adrenergic sensitivity may be part of the explanation. This study was carried out to investigate the effect of hypoglycemia on β-adrenergic sensitivity. Ten healthy male subjects (age 19–23 years) gave informed consent to take part in the study. They were hospitalized overnight at the University Hospital of Tromsø, Department of Clinical Research, on two occasions. Isoprenaline and metoprolol sensitivity tests were performed the morning after hospitalization: once after an intravenous (iv) injection of placebo (0.9% NaCl), and once after an iv injection of insulin (0.15 IU insulin/kg body weight) to induce hypoglycemia. The dose of isoprenaline needed to increase heart rate (HR) by 25 beats per minute (bpm) (I₂₅), and the dose of metoprolol (M_–12.5) needed to inhibit I₂₅ with 50% or 12.5 bpm, when injected simultaneously, were used as determinants of isoprenaline and metoprolol sensitivity. In this study, there was a significant ( p < 0.05) increase both in I₂₅ and M_–12.5 after hypoglycemia. The dose-response curve of isoprenaline/HR was significantly shifted to the right after hypoglycemia. This study shows that acute hypoglycemia induces a reduction in β-adrenegic sensitivity, and it supports the hypothesis of reduced β-adrenergic sensitivity as an important pathophysiological mechanism in hypoglycemia unawareness in IDDM.     

Influence of Cardiac Dysfunction on Fast Sodium Current Regulation by Forskolin

There are several reports of an altered β-adrenergic pathway in heart failure. Since the fast cardiac sodium current (I_NA+) is also subject to β-receptor dependent regulation, we investigated its regulation in a model of cardiac dysfunction. Adenylyl cyclase was stimulated directly with forskolin as one step in the β-adrenergic pathway. Twelve-week-old Wistar rats were infarcted by ligation of the left anterior descending coronary artery. Eight weeks later, the induced hemodynamic changes were evaluated. The left ventricular end-diastolic pressure (LVEDP) was used as a measure of the hemodynamic effects of the myocardial infarction. With the loose patch clamp technique, I_Na+ was measured in intact papillary muscles at an external sodium concentration of 150 mmol/L. Potential dependent availability was tested with pulses to 0 mV from various conditioning potentials. In animals with minor infarction (n = 7, LVEDP = 7.7 ± 0.9 mmRgj, forskolin (3 mmol/L) increased the maximal available I_Na+ to 109%± 13% of baseline values. This increase was nearly the same in the group with significant infarctions (n = 7, LVEDP = 15.7 ± 1.6 mmHg) to 113%± 6%. Thus, although we previously observed a reduction of the isoproterenol induced increase of I_{NA +} in rats with significant myocardial infarctions, this increase remalns the same when adenylyl cyclase is stimulated directly. This is consistent with a direct β-receptor down-regulation or desensitization.     

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

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

NG-Nitro-L-arginine methyl ester: a muscarinic receptor antagonist?

Summary— The effect of the nitric oxide synthase inhibitor N ^G-Nitro-L-arginine methyl ester (L-NAME) towards muscarinic receptors was studied in vitro and in vivo. L-NAME displaced the antimuscarinic ligand [³H]quinuclidinyl benzilate (³H]QNB) from its specific binding sites in rat cerebral cortex and cerebellum homogenates with a more than 10,000 fold lower affinity than atropine, pirenzepine and AFDX 116. Data for L-NAME binding were best fit according to a two-site model ( K _d 7.2 nM and 3,000 nM) in the rat cerebellum, whereas in rat cortex a one-site model ( K _d 1670 nM) was superior. In anesthetized rats and rabbits L-NAME (7.5–185 μmol/kg) attenuated a hypotensive response to Acetyl β-methyl-choline (Ac β-Me Ch)(6.25 nmol/kg) in a dose related fashion, but this effect was negligible as compared to that of atropine (8.8 and 17.7 nmol/kg). Furthermore, the effect of L-NAME was not specifically antimuscarinic since its attenuating effect against ATP- or histamine-induced responses was not statistically different from that of Ac β-Me Ch. A vagus stimulation induced bradycardia was entirely uninfluenced by L-NAME (37 μmol/kg). In isolated bladder experiments (rabbit) we demonstrated a complete lack of efficacy of L-NAME against Ac β-Me Ch induced contractions. In the pithed rat preparation L-NAME was ineffective against the McN-A-343 induced pressor responses. In summary, we demonstrated that the nitric oxide synthase inhibitor L-NAME shows very weak affinity at M₁- and M₂-receptors in the rat brain in vitro, but appears to have no significant antimuscarinic properties against M₁-, M₂- and M₃-receptor mediated effects in rats and rabbits in vivo.     

Myocardial β-adrenergic reactivity in volume overload-induced cardiac hypertrophy in the rat

Summary— The purpose of this study was to evaluate the changes in myocardial β-adrenergic reactivity in animals undergoing a 4 week cardiac volume overload. Aortocaval shunt (ACS) or sham operation (sham) were performed in male Wistar rats, and 4 weeks later, isoproterenol dose-effects (chronotropic, inotropic and lusitropic properties) were studied after pithing. Noradrenaline (NA) and adrenaline (A) concentrations and NA turn-over index were evaluated in plasma and heart ventricles, while β-adrenoceptor characteristics in ventricle homogenates and slices with [¹²⁵I]iodocyanopindolol, and the β(1)/β(2)-adrenoceptor ratio were estimated. Four weeks of cardiac volume overload resulted in a 55% increase in ventricle weight/body weight ratio (from 2.5 ± 0.1 to 3.9 ± 0.1 mg/g in sham and ACS rats, respectively) and a 20% increase in protein contents (from 11.3 ± 0.7 to 13.8 ± 1.1 mg/100 mg ventricles in sham and ACS rats, respectively). Furthermore, NA and A concentrations and NA turn-over index were increased in ACS rats (14, 40 and 80% versus sham, respectively). A shift to the right of the responses in heart rate, left ventricular systolic pressure, +d P /d t _max and -d/ P /d t _max responses following increasing doses of isoproterenol was observed, without change in the dose inducing maximum effect. Total β-adrenoceptor characteristics and β(1)/β(2) ratio were unchanged. However, β(1)-adrenoceptor density increased in epicardium while decreasing in endocardium of left ventricle from ACS rats. Rightward shift at lower doses of isoproterenol-induced cardiac responses in volume-overloaded rats are not likely due to overall β-adrenoceptor density changes.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Gamma-interferon inhibits Pneumocystis carinii attachment to lung cells by decreasing expression of lung cell-surface integrins

Pneumocystis carinii (PC) is a leading cause of pneumonia in immunocompromised patients. Previous work has shown that fibronectin (Fn) and Fn-binding integrins mediate PC attachment to lung cells in vitro . Gamma-interferon (γ-IFN) is a major factor in host defence against PC infection. To determine the effect of γ-IFN on PC attachment to lung cells, the alveolar epithelial cell line A549 was incubated with γ-IFN (0–10⁴ U mL⁻¹) and attachment of ⁵¹Cr-labelled PC to the A549 cells was quantified. PC attachment was significantly decreased ( P  < 0.01) by addition of γ-IFN with no evidence of injury to either the PC or A549 cells. Effects of γ-IFN on PC attachment were observed after 24 h and reached a maximum after 48 h of incubation. To investigate the mechanism of this decrease, we examined integrin expression on γ-IFN-treated A549 cells. A549 cell expression of the α₅ and β₁ integrin subunits was decreased, whereas expression of the α_v subunit was unchanged. Northern blot analysis showed a similar decrease in mRNA for the α₅ and β₁ integrins. Therefore, γ-IFN-mediated inhibition of PC infection may, in part, result from inhibition of PC attachment to alveolar epithelial cells caused by γ-IFN-induced decreases in alveolar integrin expression.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Alzheimer amyloid β-peptides exhibit ionophore-like properties in human erythrocytes

Abstract. There is growing evidence that the amyloid β-peptide (β₁_₄₀) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta₁_₄₀ has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether β₁-₄₀ (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2h and 4h at 37°C together with 6μmolL^-1 of β₁-₄₀ or of fragments β₁_₂₈and β₂₅-₃₅, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05μmolL^-1). Moreover, β₁_₄₀ and its two fragments induced a significant alteration of ⁴⁵Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of β₁_₄₀ and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca²⁺-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Differences in β-adrenergic receptor density and adenylate cyclase activity between normal and leukaemic leukocytes

Abstract. An identical class of high-affinity binding sites for the ¹²⁵I-labelled β-adrenergic antagonist hydroxybenzylpindolol, was identified on intact human normal and leukaemic peripheral blood leukocytes. On normal unfractionated lymphocytes, polymorphonuclear leukocytes, and monocytes, receptor density did not differ significantly (1200–1400 receptors per cell; P > 0.3), but it was higher on B- than on T-lymphocytes ( P < 005). In leukaemia, monocytic blast cells expressed highest receptor numbers, whereas very low receptor density was seen on the pathologic B-cells from chronic lymphocytic leukaemia. Among normal leukocytes, adenylate cyclase activation by hormones (isoproterenol, prostaglandin E₁, histamine) and sodium fluoride was strongest in plasma membranes from monocytes, but very weak in polymorphonuclear leukocytes either due to uncoupling of hormone receptors from adenylate cyclase or to low catalytic activity. In T-cells, enzyme activity was significantly lower than in B-cells. Loss of adenylate cyclase sensitivity to hormones and fluoride occurred in leukaemic cells from chronic and acute lymphocytic leukaemia.     

Urodilatin (Ularitide, INN): a potent bronchodilator in asthmatic subjects

Abstract. Atrial natriuretic peptide (CDD/ANP-99–126) has been identified as a bronchodilator in various species including humans. We investigated the effect of urodilatin (CDD/ANP-95–126) in 18 clinically stable asthmatics showing an increase of the FEV₁ by ≥15% after salbutamol inhalation. Prior to the study inhaled β₂-agonists were withheld for 8 h. After baseline measurements of lung function parameters (FEV₁, VC, PEF, MEF₇₅, MEF₅₀, MEF₂₅), blood pressure, and heart rate in intravenous infusion of 20, 40 or 60 ng kg^-1 min^-1 urodilatin was administered for 40min in the morning. All measurements were repeated every 10 min during the infusion, for 30 min thereafter, and after the inhalation of l.25 mg salbutamol. Forty and 60 ng kg^-1min^-1 urodilatin showed a significant effect on the central (FEV_l, PEF, MEF₇₅) and peripheral airways (MEF₅₀, MEF₂₅) after 10 min infusion ( P <0.05). A broncho-dilation not significantly different from l.25 mg salbutamol was induced by 40 ng kg^-1 min^-1 in the central airways only, while 60 ng kg^-1 min^-1 led to a similar effect at all levels of the bronchial tree. Lung function parameters returned to baseline within 30 min after cessation of the urodilatin infusion. Heart rate showed a tendency to increase after 40min infusion ( P <0.05), but blood pressure did not change significantly. In conclusion, the maximal bronchodilating effect of intravenous urodilatin in clinically stable asthmatics was comparable to l.25 mg salbutamol.     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Potentiation of platelet activation through the stimulation of P2X1 receptors

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

Muscarinic and antimuscarinic activity of acetamides related to oxotremorine in the guinea pig urinary bladder

Eight acetamide analogs of oxotremorine, shown previously to be full or nearly full muscarinic agonists on the isolated guinea pig ileum, were investigated for muscarinic activity on muscle strips of guinea pig urinary bladder. Several compounds demonstrated pronounced organ selectivity when compared to carbachol by being partial agonists or antagonists on the bladder. For example, oxotremorine and BM 34, both potent, full agonists on the ileum, were partial agonists, the latter producing less than one-half the maximum response of carbachol. BM 5 elicited no significant response, but instead was a potent antagonist to carbachol. Schild analysis with BM 5 and BM 61 indicated no muscarinic receptor heterogeneity between the bladder and ileum. Also dissociation constants of agonists and partial agonists generally agreed with those determined previously on the ileum. Furthermore, the relative efficacy of each agonist appeared to be similar in the two tissues, confirming the homogeneity of muscarinic receptors in the bladder and ileum with respect to the compounds studied. Compounds having high affinity and low intrinsic efficacy, e.g., BM 5, thus may stimulate contractile responses on the ileum and block responses on the bladder and therefore display tissue selectivity without discriminating between tissue receptors. It is suggested that this selectivity is derived from a smaller effective receptor reserve for muscarinic agonists in the bladder.     

Migraine Is Not a Platelet Disorder

SYNOPSIS
The platelet theory of migraine causation predicts that drugs inhibiting platelet activation will be effective in migraine prevention, but the literature indicates that this is only partly the case. Conversely, therapy achieving clinical benefit should be associated with reduced platelet activity. To test this concept, the β-adrenergic blockers propranolol (non-selective), metoprolol (β₁-selective) and Li-32468 (β₂-selective) were used in migraine therapy with assessments of platelet aggregation and release, plasma thromboxane A₂ (measured as thromboxane B₂) and clinical response.
Between propranolol and Li-32468, there was lack of correlation of clinical with platelet effects. Propranolol and metoprolol, whose established efficacy in migraine prophylaxis was reflected here, actually had opposite effects on platelet activity, which was increased with the former and inhibited by the latter. Yet both drugs gave elevated thromboxane B₂ levels.
In view of this complete dissociation between drug effects on platelets of migraineurs and symptoms of migraine, the platelet theory of migraine causation is untenable.     

Excitatory and inhibitory purinergic control of neck muscle nociception in anaesthetized mice

Tension-type headache is associated with noxious input from neck muscles. Due to the importance of purinergic mechanisms in muscle nociception, experimental studies typically inject α,β-methyleneadenosine 5'-triphosphate (α,β-meATP). In contrast to native adenosine 5'-triphosphate (ATP), α,β-meATP has a narrow receptor profile and remains stable in tissue. The present study administered α,β-meATP or ATP in semi-spinal neck muscles in anaesthetized mice ( n  = 65) in order to address different effects in neck muscle nociception. The jaw-opening reflex monitored the impact of neck muscle noxious input on brainstem processing. Injection of α,β-meATP induced reflex facilitation in a dose-dependent manner. In contrast, only the lowest ATP dosage evoked facilitation. Preceding P2Y₁ receptor blockade revealed facilitation even under high-dosage ATP. Ongoing facilitation after α,β-meATP injection neutralized under subsequent activation of P2Y₁ receptors. Results demonstrate opposing excitatory P2X and inhibitory P2Y effects of ATP in neck muscle nociception. These mechanisms may be involved in the pathophysiology of neck muscle pain in man.     

Direct determination of acetylcholine release by radioimmunoassay and presence of presynaptic M1 muscarinic receptors in guinea pig ileum

Radioimmunoassay for acetylcholine (ACh) with a sensitivity of 10 pg/tube was applied to the direct determination of ACh output from the nerve endings in longitudinal muscle strips of guinea pig ileum. The strips were preincubated with an irreversible cholinesterase inhibitor and superfused with Krebs' solution under various experimental conditions. Pirenzepine (0.1-10 microM) and atropine (10-100 nM) produced an increase in electrically evoked ACh output through the inhibition of presynaptic muscarinic receptors. Contractile response to endogenous ACh released by electrical stimulation was enhanced by pirenzepine and atropine at lower concentrations, whereas the highest concentrations of pirenzepine (10 microM) and atropine (100 nM) caused a reduction in the enhanced contractile response and a significantly diminished response, respectively. These results demonstrate that the concentrations of pirenzepine and atropine, effective in inhibiting presynaptic muscarinic receptors, differ from those inhibiting postsynaptic muscarinic receptors and suggest the possibility that presynaptic M1 muscarinic receptors regulating ACh output may be present in the guinea pig ileum.