α1-adrenoceptor subtype(s) mediating noradrenaline-induced contractions of the guinea-pig aorta

Summary— The effect of α₁-adrenoceptor subtype selective antagonists, WB 4101, SZL-49 and chloroethylclonidine on noradrenaline-induced contractions of the guinea-pig aorta has been studied in an attempt to identify the α₁-adrenoceptor subtype(s) involved in the response. Noradrenaline and SDZ NVI 085 induced contractions of the aorta. Noradrenaline-induced contractions were competitively antagonised by WB 4101 (pA₂ = 8.92, slope = 1.05). The contractions were significantly reduced by SZL-49 but not by chloroethylclonidine, indicating an action on α_1A-adrenoceptor subtype. Noradrenaline-induced contractions of the aorta were not inhibited by nifedipine (10⁻⁶ M). The results are interpreted to suggest that α_1A-adrenoceptor subtype mediates noradrenaline-induced contractions of the guinea-pig aorta and that activation of α_1A-adrenoceptor subtype in the guinea-pig aorta is probably linked to intracellular Ca⁺⁺ release.     

α1 and α2-adrenergic control of large and small coronary arteries during exercise in conscious dogs under β-blockade

Summary— The aim of this study was to determine the relative roles of α₁-and α₂-adrenoceptors at the level of large epicardial and small resistance coronary arteries when sympathetic tone is increased by exercise in conscious dogs. The responses of left circumflex coronary artery diameter and blood flow were investigated at rest and during graded treadmill exercise (5, 10 and 12 km/h) in six chronically instrumented dogs during control conditions (saline) and after administration of propranolol (1 mg/kg) either alone or in combination with either prazosin (50 μg/kg), or idazoxan (300 μg/kg), or the association of prazosin + idazoxan (same doses). In control conditions, graded treadmill exercise resulted in a progressive increase in coronary artery diameter (+ 3.8 ± 0.6% from 3479 ± 80 μm) and in a decrease in coronary vascular resistance (- 46.0 ± 4.5% from 8.49 ± 1.51 mmHg/cm/s). Propranolol significantly constricted large (- 4.4 ± 0.6% from 3486 ± 87 μm) and limited dilation of small coronary arteries during exercise. These coronary effects of propranolol remained unchanged following additional α₂-adrenoceptor blockade by idazoxan but were abolished following α₁-adrenoceptor blockade by prazosin, given either alone or combined with idazoxan. Thus, α₁- but not α₂-adrenoceptors are responsible for propranolol-induced constriction of large coronary arteries and limitation of small coronary arteries dilation during exercise in conscious dogs.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

Effects of α1-acid glycoprotein in different rodent models of shock

Summary— It was the aim of the present study to investigate the effects of the acute phase protein α₁-acid glycoprotein in different models of shock. The human plasma preparation used was without effect on mortality in lipopolysaccharide-injected mice when administered in two different doses (1 or 0.33 g/kg iv) and according to different treatment schedules. The same preparation significantly increased survival rate (48 h) in rats with septic peritonitis. This effect was seen when α₁-acid glycoprotein (200 mg/kg iv) was given 15 min prior to and 24 h after cecal puncture. All other dose regimes tested were without significant effect on survival rate. A hemorrhagic/hypovolemic shock model (including a defined trauma) in rats resuscitated with 200 mg/kg α₁-acid glycoprotein resulted in significantly higher values of mean arterial blood pressure, cardiac output and stroke volume when compared to corresponding values obtained after resuscitation with Ringer's solution or 200 mg/kg albumin iv (free of α₁-acid glycoprotein; placebo formulation). Taking all other possible mechanisms of α₁-acid glycoprotein into consideration, the partially protective effects of the preparation are explained by enhancing the capillary barrier function and thereby maintaining perfusion of vital organs.     

α1-adrenoceptor subtypes mediating vasoconstriction in the carotid circulation of anaesthetized pigs: possible avenues for antimigraine drug development

It has recently been shown that the alpha-adrenoceptors mediating vasoconstriction of porcine carotid arteriovenous anastomoses resemble both alpha1- and alpha2-adrenoceptors, but no attempt was made to identify the specific subtypes (alpha1A, alpha1B and alpha1D) involved. Therefore, the present study was designed to elucidate the specific subtype(s) of alpha1-adrenoceptors involved in the above response, using the alpha1-adrenoceptor agonist phenylephrine and alpha1-adrenoceptor antagonists 5-methylurapidil (alpha1A), L-765 314 (alpha1B) and BMY 7378 (alpha1D). Ten-minute intracarotid infusions of phenylephrine (1, 3 and 10 microgkg-1.min-1) induced a dose-dependent decrease in total carotid and arteriovenous anastomotic conductance, accompanied by a small tachycardia. These carotid vascular effects were abolished by L-765 314 (1000 microgkg-1; i.v.), while these responses were only attenuated by 5-methylurapidil (1000 microgkg-1; i.v.), and BMY 7378 (1000 microgkg-1; i.v.). Furthermore, intravenous bolus injections of phenylephrine (3 and 10 microgkg-1) produced a dose-dependent vasopressor response, which was only affected by 1000 microgkg-1 of 5-methylurapidil, while the other antagonists were ineffective. These results, coupled to the binding affinities of the above antagonists at the different alpha1-adrenoceptors, suggest that both alpha1A- and alpha1B-adrenoceptors mediate constriction of carotid arteriovenous anastomoses in anaesthetized pigs. In view of the less ubiquitous nature of alpha1B- compared to alpha1A-adrenoceptors, the development of potent and selective alpha1B-adrenoceptor agonists may prove to be important for the treatment of migraine.     

Beta3-adrenoceptors in the cardiovascular system

Summary— Behind the classic beta₁, and beta₂-adrenoceptors, recent molecular and pharmacological studies have described a new receptor, called the beta₃-adrenoceptor, in various mammalian tissues (brown and white adipose tissue, digestive smooth muscle). Few authors have investigated the putative existence of the beta₃-adrenoceptor in the cardiovascular system. This paper reviews the available data. In vitro studies show that beta₃-adrenoceptor agonists (BRL 37344, CGP 12177) induce a relaxation of fragments of rat carotid artery which is not antagonized by propranolol. In dogs, these drugs elicit a decrease in blood pressure due to peripheral vasodilation and an increase in heart rate which is of baroreflex origin. The vasodilating effects are mainly observed in cutaneous and adipose tissue vessels and cannot be explained by any known transductional mechanism. Activation of this vascular β₃-adrenoceptor requires higher doses of catecholamines than for β₁- or β₂-adrenoceptors. In humans, the cardiovascular effects of beta₃-adrenoceptor agonists are explained by the activation of beta₁- or beta₂ (and not beta₃-)-adrenoceptors. These studies suggest the presence of vascular (but not cardiac) beta₃-adrenoceptors in dogs. In other species, including man, the presence of such cardiac β₃-adrenoceptors remains to be resolved. Their physiological relevance remains unknown.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

alpha(1A)-adrenoceptors mediate sympathetically evoked pupillary dilation in rats.

Evidence suggests that in some species (cats, rabbits, and possibly humans) alpha-adrenoceptors in the iris dilator muscle are "atypical" in that they cannot be readily classified by conventional criteria. This study was undertaken in an attempt to characterize the alpha-adrenoceptor subtype(s) mediating sympathetically elicited mydriasis in rats. Frequency-response pupillary dilator curves were generated by stimulation of the preganglionic cervical sympathetic nerve (1-32 Hz) in pentobarbital-anesthetized rats. Evoked responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-1 mg/kg). The selective alpha(1)-adrenergic antagonist, prazosin (0.01-1 mg/kg), also was effective, although alpha(2)-adrenergic antagonism with rauwolscine (0.1-1 mg/kg) was not. alpha(1A)-Adrenoceptor-selective antagonists, 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101; 0.1-1 mg/kg) and 5-methylurapidil (0.1-1 mg/kg), as well as the alpha(1D)-adrenoceptor-selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY-7378; 1-3 mg/kg), were used to determine the subtype(s) involved. Evoked mydriasis was significantly antagonized by both WB-4101 and 5-methylurapidil but not by BMY-7378. These results suggest that, unlike some other species, adrenoceptors in the rat iris dilator mediating neurogenic mydriasis are "typical" and, in addition, can be characterized as being primarily of the alpha(1A)-adrenoceptor subtype.     

Alpha-adrenoceptors in canine mesenteric artery are predominantly 1A subtype: pharmacological and immunochemical evidence.

We wanted to determine which alpha-adrenoceptor subtypes mediate phenylephrine (PE) contraction of dog mesenteric artery in vitro. We studied antagonisms in response to prazosin, 2-(2, 6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane, 5-methylurapidil, N-[2-(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine HCl (RS 17053), 8-3-[4-(2-methoxyphenyl)-1-piperazinyl]propylcarbamoyl)-3-methyl-4 -oxo-22-phenyl-4H-1-benzopyran 2HCl [SB216469 (Rec 15/2739)], BMY 7378, 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol++ + [4,5]decane-7,9-dione HCl, MDL 72832, and 7-chloro-2-bromo-3,4,5, 6-tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine. pK(B) values for prazosin, 5-methylurapidil, MDL 72832, and RS-17053 were consistent with action on alpha(1A)-adrenoceptors but decreased with concentration. pK(B) values (9.6) for Rec 15/2739 (alpha(1L/1A)-adrenoceptor selective) were constant. Antagonism by BMY 7378, 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3, 2-ef]3-benzapine, and 8-[2-(1, 4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]de cane-7,9-dione HCl gave pK(B) values between those expected for alpha(1A)- and alpha(1D)-adrenoceptors. Chloroethylclonidine (100 microM) shifted EC(50) values for PE rightward and decreased E(max) values but left large residual responses. After 100 microM chloroethylclonidine, either BMY 7378 (100 nM) or RS-17053 (300 nM) increased EC(50) values for PE contractions with pK(B) values like those of controls. At 6 nM, phenoxybenzamine increased the EC(50) values and reduced E(max) values; prior Rec 15/2739, but not prior BMY 7378, protected receptors against inactivation. An antibody against the alpha(1B)-adrenoceptors immunostained muscle of aorta but not mesenteric artery. We conclude that dog mesenteric artery contains alpha(1A)-adrenoceptors. Discrepancies among responses expected if only these receptors are present may result from pleiotropic functional effects at this receptor and the presence of alpha(1L)-adrenoceptors.     

Anti-Migraine Drug Interactions with Cloned Human 5-Hydroxytryptamine 1 , Receptor Subtypes

SYNOPSIS
Multiple 5-hydroxytryptamine (5-HT) receptors have been identified in humans. A subgroup of these receptors (designated 5-HT ₁ receptors) have been hypothesized to be involved in the mechanism of action of acute anti-migraine drugs. At present, this hypothesis cannot be tested directly in human tissues due to technical limitations. However, recent molecular biological advances have allowed for the development of assays employing cloned human 5-HT ₁ receptors expressed in cells by DNA transfection. This study analyzed the ability of ergotamine, dihydroergotamine, 5-HT and sumatriptan to interact with the four known human 5-HT ₁ receptor subtypes. The four acute anti-migraine agents interacted with all 4 human 5-HT ₁ receptor subtypes with less than 1 μM affinity. However, drug affinities for the human 5-HT _1B and 5-HT _1D receptors correlate most closely with the rank order of clinical dosages used to treat a migraine attack. Therefore, these data indicate that human 5-HT _1B and/or 5-HT _1D receptors are likely to mediate the therapeutic efficacy of acute anti-migraine drugs.     

Effects of yohimbine, an α2-adrenoceptor antagonist, on experimental neurogenic orthostatic hypotension

Summary— Yohimbine has been proposed for the treatment of neurogenic orthostatic hypotension; however, no controlled trial has been performed in experimental models of orthostatic hypotension or in patients with autonomic failure. The aim of the present study was to compare the effects of yohimbine (0.05 mg/kg, intravenously [iv]) and placebo (saline) in a new model of neurogenic orthostatic hypotension obtained by sinoaortic denervation (SAD) in chloralose-anaesthetized dogs. Blood pressure, heart rate, noradrenaline plasma levels and systolic blood pressure and heart rate short-term variabilities (calculated on low frequency [40–50 MHz] and high frequency [390–490 MHz] bands) were measured in supine position and after a 10 min 80° head-up tilting. The drugs were administered in a double-blind cross-over randomized fashion. The head-up tilting performed in normal animals increased diastolic blood pressure (+12 ± 4 mmHg), heart rate (+39 ± 12 beats per minute [bpm]), the low frequency band of systolic blood pressure and noradrenaline plasma level, without changing systolic blood pressure or heart rate variability. In SAD dogs, a marked fall in systolic (-80 ± 11 mmHg) and diastolic (-43 ± 4 mmHg) blood pressures was observed within 1 min after placebo, without modification in heart rate, systolic blood pressure and heart rate short-term variabilities and noradrenaline plasma levels. In SAD dogs, yohimbine (0.05 mg/kg, iv) delayed the blood pressure fall elicited by head-up tilting, but failed to modify its magnitude. These results show that, in the model of orthostatic hypotension obtained by SAD, yohimbine, at an α₂-adrenoceptor selective dose (0.05 mg/kg), delays the fall in blood pressure elicited by head-up tilting. The effect of yohimbine can be explained by an increase in sympathetic tone.     

Relationship between fractional calcium absorption and gastric emptying

Abstract. The relationship between calcium absorption and gastric emptying and the precision of measurement of fractional calcium absorption using a single isotope technique were evaluated in 14 normal postmenopausal women (age range 61–72 years). On two occasions separated by between 5 and 15 days, each subject was given 250 mL water containing 0.2 MBq of ⁴⁵Ca in 20 mg of calcium carrier as the chloride, 20 mg kg^-1 paracetamol and 9 MBq of ^99mTc sulphur colloid. Venous blood samples were taken at -2, 15, 30, 45, 60, 90, 120, 150 and 180 min after consumption of the drink, and gastric emptying (GE) was monitored with a gamma camera. Fractional calcium absorption in the first hour (α₆) was calculated from the blood samples obtained at 15, 30, 45, 60, 90 and 120 min. An absorption rate was also derived from the 60 min sample using only a calibration curve (α₁). There were close correlations between radiocalcium absorption on the two study days ( r = 0.89, P < 0.001 for both α₁ and α₆) and between α₁ and α₆ ( r = 0.93, P < 0.001). Plasma paracetamol concentrations at 15 min were directly related to the early phase of GE ( r = 0.42, P < 0.05). In contrast, calcium absorption was inversely related to GE ( r — 0.45, P < 0.05). We conclude that radiocalcium absorption is not greatly influenced by gastric emptying rate and that the single blood sample procedure has similar precision to the six-blood sample test.     

Serum β2-microglobulin at remission and relapse in patients with multiple myeloma

Abstract. Serum β₂-microglobulin (S-β₂m) was determined before treatment in fifty patients with multiple myeloma (MM), twenty-two of which had pure Bence Jones (BJ) myeloma. S-β₂m was related to clinical stage before, but not after, a correction of S-β₂m values for co-existent raised S-creatinine values (> 106 μmol 1^-1)- However, S-β₂m as well as corrected β₂m was a parameter of prognostic value. The median expected survival time was 14 months at S-β₂m values > 8·0 mg 1^-1 and 56 months at values<3·5 mg 1^-1. A response to treatment was associated with a decrease of S-β₂m and a stationary course with unchanged values, whereas a relapse or progressive disease was connected with an increase. In β₂m producers', i.e. patients with a clear initial high S-β₂m, serial determinations are of value for monitoring patients with MM. In particular, this is the case in BJ myelomas, as they lack a quantifiable serum M-component. With respect to β₂m no difference was found between BJ and other patients with MM.     

Positive chronotropic activity of bradykinin in the pithed normotensive rat

Summary— The positive chronotropic effect of bradykinin was investigated in the pithed rat preparation. Cumulative treatment with bradykinin (0.20 nmol/kg-6.59 μmol/kg, intravenous [iv]) caused a dose-dependent increase in heart rate (HR) by a maximum of 80 ± 3.3 beats min^-1. In contrast, the active metabolite of bradykinin and selective bradykinin B₁- receptor agonist, [des-Arg⁹]-bradykinin did not influence the spontaneous frequency of beating. Propranolol alone reduced the bradykinin-induced increase in HR and a combination of propranolol with prazosin abolished the chronotropic effect of bradykinin. The selective bradykinin B₂ receptor antagonist, Hoe 140, dose-dependently shifted the dose-response curves of bradykinin to the right, whereas the bradykinin B1 receptor antagonist, des-Arg¹⁰-[Leu⁹]-kallidin proved ineffective. From our experiments it may be concluded that bradykinin induces tachycardia in the pithed rat primarily by stimulating the sympathetic ganglia leading to the release of noradrenaline, which subsequently activates cardiac β₁-adrenoceptors. The bradykinin-induced chronotropic effect is mediated by bradykinin B₂-receptors, whereas B₁-receptors appear not to be involved.     

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.     

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.     

Effects of interleukin-10 and modulators of cyclic AMP formation on endotoxin-induced inflammation in rat lung

Abstract— The aim of this study was to investigate the effects of IL-10, a cell permeable analogue of cyclic AMP, dibutyryl-cAMP (db-cAMP), modulators of intracellular cyclic AMP such as phosphodiesterase (PDE) inhibitors and a β₂-adrenoceptor agonist, salmeterol, on pulmonary inflammation following acute lung injury induced by endotoxin exposure in rats. Pulmonary inflammation was induced in adult Wistar rats by a 60-min exposure to endotoxin (lipopolysaccharide, LPS, 100 μg/mL). 4 h later bronchoalveolar lavage (BAL) was performed. The PDE inhibitors, rolipram (3 and 5 mg/kg) and theophylline (30 and 100 mg/kg) inhibited neutrophil recruitment, TNF-α release and cellular activation in BAL. Salmeterol (0.5 mg/mL) and IL-10 (0.1 μg) only inhibit TNF-α increase in the BAL fluid and db-AMPc (2.5 μg/rat) was ineffective. The present data show that the selective PDE4 inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline, markedly reduced the pulmonary inflammation associated with acute lung injury in the rat. These effects may be mediated in part by IL-10 rather than by cyclic AMP, as demonstrated by the potent inhibitory activity of exogenous IL-10 on the increase in TNF-α release in BAL fluid of rats exposed to LPS.     

Effects of the 5-hydroxytryptamine receptor antagonist, BMY 7378, on 5-hydroxytryptamine neurotransmission: electrophysiological studies in the rat central nervous system

In the present electrophysiological studies, the effects of the putative 5-hydroxytryptamine (5-HT) receptor antagonist, BMY 7378, on the response of dorsal raphe nucleus 5-HT neurons and of CA3 dorsal hippocampus pyramidal neurons to 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were investigated in chloral hydrate-anesthetized rats. The effectiveness of microiontophoretically applied 5-HT and 8-OH-DPAT in suppressing the firing activity of both neuronal populations was assessed before and during the microiontophoretic application of BMY 7378. BMY 7378 reduced the effectiveness of 8-OH-DPAT, but not that of 5-HT, in depressing 5-HT neuron firing rate, whereas that of both agonists was reduced by concurrent application of BMY 7378 in the hippocampus. To assess whether endogenous 5-HT also could be antagonized, the response of pyramidal neurons to electrical activation of the 5-HT pathway was determined before and after i.v. BMY 7378. Low doses enhanced the efficacy of the stimulation, whereas higher doses decreased it. The latter finding suggests that BMY 7378 antagonizes the effect of endogenous 5-HT. Three procedures were used to investigate the enhancing effect of BMY 7378 on 5-HT synaptic transmission: 1) administration of BMY 7378 after terminal 5-HT autoreceptor blockade by methiothepin: methiothepin abolished the enhancing effect of BMY 7378; 2) blockade of the effect of RU 24969, a terminal 5-HT autoreceptor agonist: pretreatment with methiothepin, but not with BMY 7378, blocked the effect of RU 24969 on 5-HT synaptic transmission; and 3) administration of BMY 7378 during a reduced level of activation of terminal 5-HT autoreceptors, obtained by lowering the stimulation frequency from 1 to 0.5 Hz: the enhancing effect of BMY 7378 was reduced when the stimulation was delivered at 0.5 Hz. It is concluded that BMY 7378 is an effective antagonist of 5-HT1A receptors in vivo and that the mechanism of its enhancing effect on 5-HT transmission at low doses, although still undetermined, is not due to a competitive interaction at the terminal 5-HT autoreceptor.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.