Abstracts of papers presented at the XIVth Platelet Meeting, 15-17 October 1999, Castle Oppurg,Thuringia, Germany

We report here a study of platelet aggregation in diabetes, induced by epinephrine and its inhibition by yohimbine hydrochloride (YH), an α₂-adrenergic receptor-blocking agent. Interestingly, emergence of spontaneous platelet macroaggregation (SPMA) was observed in six out of 75 cases in the absence of any agonist. The SPMA cases were strongly associated with insensitivity to YH (in contrast with non-SPMA cases) when epinephrine was used as an agonist. We suggest that the observed correlation is a result of over expression of platelet α₂-adrenoceptors in such subjects. The quantitative nature of the effect is supported by the observation that addition of YH at higher concentration (more than 5?µM) led to restoration of the adrenergic receptor-blocking activity of the said agent. Eventually for non-SPMA subjects YH exhibited blocking activity even at lower concentration. The aggregation profile and the platelet morphology of the SPMA cases had distinctive features as compared to microaggregates formed in other diabetic subjects (non-SPMA cases).     

Venoconstrictor responses to dihydroergocristine and dihydroergotamine: Evidence for the involvement of 5-HT1 like receptors

Summary Dihydroergocristine (DHEC) and dihydroergotamine (DHE) were investigated on canine saphenous veins in vivo and on canine saphenous veins and basilar arteries in vitro. Following local IV infusion in vivo, the venoconstrictor response to DHEC was about 30% weaker than that produced by DHE. When administered orally, however, both ergot alkaloids elicited similar venoconstrictor effects. In vitro maximal contractile responses to DHEC and DHE of basilar arteries were only 20–30% of those produced by 5-HT, whereas in saphenous veins both DHEC and DHE elicited similar maximal effects as those observed with 5-HT. In saphenous veins, methiothepin antagonized venoconstrictor responses to 5-HT, DHEC, and DHE within the same concentration range, being significantly less potent when tested against noradrenaline. The reverse was true for yohimbine, which was significantly more potent against noradrenaline than againificantly more potent against noradrenaline than against 5-HT, DHEC, and DHE. It is suggested that the venoconstrictor responses to both DHEC and DHE are mediated through 5-HT₁-like receptors.     

The effect of clonidine injected centrally on serum-free fatty acids and glucose concentration in the rat

Clonidine administered intracerebroventricularly (ICV) to conscious rats induced a significant dose-dependent increase in serum free fatty acid (FFA) and blood glucose levels. The rise in FFA and glucose concentrations after a dose of 10 μg averaged 80%–100% of the control levels. The α-adrenoceptor antagonists yohimbine, phentolamine, and phenoxybenzamine, administered alone ICV, did not substantially influence the control FFA and glucose levels. A hyperlipemic response to clonidine (10 μg) was abolished by an ICV pretreatment with yohimbine and was strongly antagonized by phentolamine but not by phenoxybenzamine. The clonidine-induced hyperglycemia was significantly diminished by phentolamine and only partly by yohimbine, whereas phenoxybenzamine was ineffective here. The histamine H₂-receptor antagonist cimetidine, given ICV, failed to change these effects of clonidine. It seems likely that clonidine, given ICV, induces hyperlipemic and hyperglycemic effects through the stimulation of central α-adrenoceptors but not histamine H₂-receptors. The lipolytic action of the drug depends more on the activation of α₂-adrenoceptors.     

Cerebrospinal fluid corticotropin-releasing hormone in healthy humans: effects of yohimbine and naloxone

CRH neurons projecting from the paraventricular nucleus (PVN) of the hypothalamus to the median eminence control hypothalamic-pituitary-adrenal (HPA) axis activity. However, CRH neurons outside the PVN as well as PVN neurons projecting to sites other than the median eminence also contribute to the stress response and may play a role in mood and anxiety disorders. We have attempted to investigate possible noradrenergic and opioid regulation of these non-HPA CRH neurons. We hypothesized that yohimbine (an alpha2-adrenergic antagonist) would have stimulatory action on non-HPA CRH neurons, whereas naloxone (a mu-opioid receptor antagonist) would not have this effect. Adult normal volunteers received i.v. yohimbine (n = 5; 0.4 microg/kg), naloxone (n = 4; 125 microg/kg), or placebo (n = 3; 0.9% saline). Cerebrospinal fluid (CSF) was collected continuously, and concentrations of CSF CRH, CSF norepinephrine (NE), and plasma cortisol were measured. Administration of either yohimbine or naloxone caused significant increases in plasma cortisol concentrations over time. Although yohimbine robustly increased CSF NE levels and appeared to increase CSF CRH levels, these effects were not seen after naloxone or placebo administration. Intraindividual correlations were not observed between the measured concentrations of plasma cortisol and CSF CRH for any of the subjects. The results support the idea that CSF CRH concentrations reflect the activity of non-HPA CRH neurons. Although both yohimbine and naloxone stimulated the HPA axis, only yohimbine appeared to have stimulatory effects on central NE and non-HPA CRH.     

Identification of vascular postsynaptic alpha 1- and alpha 2-adrenoceptors in man

We studied postsynaptic alpha-adrenoceptors in human blood vessels by measuring the influence on forearm blood flow induced by intra-arterial infusions of selective alpha 1- and alpha 2-adrenoceptor agonists (methoxamine, B-HT 933, clonidine and guanfacine) and antagonists (doxazosin and yohimbine). The studies were done in healthy volunteers, and forearm blood flow was measured by plethysmography. All agonists produced a significant and dose-dependent vasoconstriction. The effect of B-HT 933 was completely abolished by the concomitant infusion of yohimbine, whereas it was hardly influenced by doxazosin. The effect of methoxamine was prevented by doxazosin and little influenced by yohimbine. The vasoconstriction by clonidine and guanfacine was partially prevented by both doxazosin and yohimbine. The single intra-arterial infusion of yohimbine, as well as doxazosin, resulted in vasodilation. These findings provide strong evidence for the existence of postsynaptic alpha 1- as well as alpha 2-adrenoceptors, both mediating vasoconstriction and contributing to basal vascular tone. The (patho-)physiological significance of this subdivision of alpha-adrenoceptors remains to be elucidated.     

Central alpha 2 adrenoceptor-mediated pancreatic exocrine response to yohimbine in anesthetized dog

Effects of yohimbine on the secretion of pancreatic juice in anesthetized dogs were investigated. Intravenous yohimbine (0.1-1 mg/kg) caused dose-dependent increases in the secretion of pancreatic juice and vasodilation. However, intra-arterial yohimbine (0.3-1 mg) did not cause any secretory responses. The secretory activity of 0.3 mg/kg of yohimbine was approximately equal to that of 0.04 U/kg of secretin and 40 mg/kg of 2-deoxy-D-glucose (2-DG). Secretory responses to yohimbine were inhibited by pretreatment with clonidine or atropine and abolished by vagotomy. The concentration of protein in the pancreatic juice induced by yohimbine was increased, but the bicarbonate concentration was scarcely changed. These results indicate that yohimbine stimulates, at least in part, pancreatic exocrine secretion by acting on the central alpha 2 adrenoceptor in the dog.     

Contractile action of yohimbine in the dog basilar artery

Yohimbine (10(-8) to 10(-4) M) produced a concentration-dependent contraction of the isolated dog basilar artery. The maximal amplitude of the contraction induced by yohimbine was approximately 50% of that induced by 5-hydroxytryptamine (5-HT) and twice as large as that of noradrenaline. This response to yohimbine (3 X 10(-7) M) was attenuated by 5-HT receptor antagonists such as 5-methoxygramine, methysergide and ketanserin. Phentolamine also antagonized the yohimbine response but prazosin and DG-5128 did not. The 5-HT antagonists and phentolamine produced similar inhibition of the response to 5-HT. Propranolol, atropine and diphenhydramine did not modify the contractile responses to yohimbine and 5-HT. These results suggest that the contractile response to yohimbine is mediated through 5-HT receptors. In addition to the contractile action, yohimbine at concentrations over 10(-6) M inhibited the contractile response to 5-HT. Thus, the possibility that yohimbine is a partial agonist on 5-HT receptors in the dog basilar artery has to be considered.     

Effects of yohimbine on human sympathetic nervous system function

The alpha 2-adrenergic receptor antagonist yohimbine is often used as a neuroendocrine probe in human studies, in which it is assumed to increase plasma norepinephrine (NE) by increasing sympathetic outflow. In this study we have tested that assumption by using a radioisotope dilution technique to measure norepinephrine (NE) kinetics in arterialized plasma after administration of oral yohimbine (20 or 40 mg) or placebo to normal young men. In agreement with previous studies, we found that yohimbine causes dose-dependent increases in blood pressure, heart rate, and plasma NE. We further found that the increase in plasma NE is, in fact, due to an increase in the rate of appearance of NE into plasma and not to reduced NE clearance from plasma. In addition, we found that yohimbine causes a dose-dependent increase in plasma epinephrine, which had not been found in studies measuring catecholamines in venous plasma. We conclude that yohimbine increases plasma NE levels by increasing the rate of NE release from sympathetic nerves, and probably increases epinephrine release from the adrenals.     

Plasma catecholamine levels and lipid mobilization induced by yohimbine in obese and non-obese women

Oral yohimbine administration (0.2 mg/kg) induced lipid mobilization (increase in plasma non-esterified fatty acids, NEFA) in fasting non-obese women (body mass index BMI = 20.2 +/- 0.5, age 35.5 +/- 2.7 years) without significant action on plasma glucose, insulin levels, heart rate or blood pressure during the time-course of the experiment (240 min). Plasma norpinephrine (but not epinephrine) concentrations were increased (100 percent) after oral yohimbine administration. Oral administration of propranolol (40 mg, 60 min before yohimbine) reduced the lipid-mobilizing action of yohimbine (70 percent) during the 60 min following its administration and then totally suppressed its effect until the end of the experimental period (180 min). In fasting obese women (BMI = 36.4 +/- 2.1, age 37 +/- 3.6 years), yohimbine provoked an increase in plasma NEFA levels which was not markedly different from that observed in non-obese subjects. It had no significant effect on plasma glucose, insulin levels, heart rate or blood pressure. Plasma norepinephrine increased in the same proportions. The lipid-mobilizing effect of yohimbine in women is mainly attributable to the increase in synaptic norepinephrine with a resultant increment in lipolysis by beta-adrenergic agonism. In the standard fasting conditions (12 hours) the blockade of the antilipolytic fat cell alpha 2-adrenoceptors seems to be a minor component of the lipomobilizing effect of yohimbine. Morever, when compared with non-obese women, the lipomobilizing effect of yohimbine is not enhanced in obese women.     

Yohimbine Can Induce Ethanol Tolerance in an in Vitro Preparation of Rat Locus Coeruleus

Noradrenergic neurons have been implicated in the development of ethanol dependence and tolerance. Moreover, the development of an hyposensitivity of alpha 2 adrenoceptors has been postulated during long-term exposition to ethanol. In order to test the putative role of alpha 2 receptors in ethanol intoxication, we have studied the interaction between ethanol and yohimbine, an alpha 2 antagonist, on the spontaneous firing rate of rat locus coeruleus (LC) in an in vitro slice model. The spikes from single neurons were recorded by glass microelectrodes. Ethanol at 100 mM, a concentration that parallels the behavioral effects in the human and in the animals, inhibits the firing activity of some LC cells. This inhibition was quickly reversed after stopping the ethanol perfusion and was observed for each further administration. However, if yohimbine (20 microM) was simultaneously perfused, the ethanol-induced inhibition was rapidly antagonized. This effect is reversible after long time washout of yohimbine. This suggests that alpha 2 adrenoceptors could be implicated in the inhibitory effect of ethanol on LC noradrenergic neurons and perhaps in the development of tolerance. However, other hypotheses are discussed, because yohimbine can also antagonize other types of receptors.     

Antiarrhythmic effects of alpha-adrenoceptor antagonists in guinea pig ventricular myocardium

Antiarrhythmic effects of alpha-adrenoceptor antagonists were assessed in the reserpinized guinea pig ventricular myocardium. Both bunazosin (1 to 3 x 10(-7) M), a new alpha 1-adrenoceptor antagonist, and yohimbine (1 to 3 x 10(-7) M), another adrenoceptor antagonist, suppressed the transient depolarization and triggered activity induced by a train of rapid stimuli in the solution containing low potassium ion (K+), high calcium ion (Ca2+) and strophanthidin (1 to 5 x 10(-7) M). Bunazosin (3 x 10(-6) M) abolished the facilitatory effect of hypoxia on beta-adrenoceptor mediated abnormal automaticity. To clarify the mechanisms underlying the antiarrhythmic properties of alpha-adrenoceptor antagonists, their electrophysiologic effects on the fast and slow action potentials were investigated. Alpha-adrenoceptor antagonists (bunazosin, yohimbine and phentolamine) suppressed the slow response in a dose-related manner. The voltage-dependent block and use-dependent block of the maximal rate of rise (Vmax) of action potentials by bunazosin (10(-5) to 10(-4) M) and yohimbine (10(-6) to 10(-5) M) were studied. The analysis of the onset and recovery kinetics from the use-dependent block of drugs showed that both bunazosin and yohimbine act as slow kinetic drugs. It is concluded that alpha-adrenoceptor antagonists seem to have an antiarrhythmic effect through the inhibition of fast sodium ion (Na+) and slow Ca2+ currents of the cell membrane independently of blockade of myocardial alpha-adrenoceptors.     

Pancreatic islet circulation in relation to the diabetogenic action of streptozotocin in the rat

A possible relationship between pancreatic islet blood flow (IBF) and the diabetogenic action of streptozotocin (STZ) has been investigated in rats injected ip with saline, glucose, propranolol, phentolamine, yohimbine, or adrenaline. A low dose (25 mg/kg BW) of STZ was given iv 10 min later, and serum glucose concentrations were determined after 3 and 7 days. Measurements of IBF were performed in separate experiments with a microsphere technique 10 min after injection of the drugs. Administration of phentolamine, glucose, or yohimbine was followed by overt diabetes, whereas animals injected with saline, adrenaline, or propranolol failed to become diabetics. Blood flow measurements showed an increase in IBF 10 min after the injection of glucose or propranolol, while there were no significant effects of the other drugs. It is concluded that changes in IBF are of minor importance for the action of agents modulating the diabetogenic effect of STZ. However, the potentiated hyperglycemic effects by glucose, phentolamine, or yohimbine in combination with STZ seemed to coincide with increased insulin secretion, as reflected by elevated serum insulin concentrations at the time of STZ administration.     

Genetic variation at the human alpha2B-adrenergic receptor locus: role in blood pressure variation and yohimbine response

Exaggerated response to alpha2-adrenergic receptor (alpha2-AR) blockade by yohimbine in normotensive subjects is an intermediate phenotype that predicts increased risk for development of hypertension. Here, we assessed the 3 alpha2-AR loci (alpha2A, alpha2B, alpha2C) as candidate genes for their influence on baseline and yohimbine-mediated increase in mean arterial pressure. Because initial results with 173 individuals implicated a possible association of yohimbine response with genetic variation at a site in the alpha2B-AR gene, but not at sites in the other 2 alpha2-AR, we sequenced the alpha2B-AR gene (4.4 kb, including 1.2 kb upstream and 1.9 kb distal to the coding sequence) in those subjects and an additional 81 individuals to search for other alpha2B-AR variants. We identified 25 polymorphisms, of which 14 are previously unreported, and 2 major haplotypes that differ by the presence/absence of a 9-bp in-frame deletion that encodes Glu301 to Glu303. Frequency differences in haplotypes were observed between blacks and whites but did not predict response to yohimbine. Genotyping of 2 additional white cohorts, including 1269 individuals with extremes in blood pressure selected from >50,000 subjects, also failed to reveal an association of the 2 major alpha2B-AR haplotypes with differences in blood pressure. Thus, despite considerable polymorphism in alpha2-AR genes, such variation is not a major determinant of variability in yohimbine response and by inference, in susceptibility to essential hypertension.     

Yohimbine increases cerebrospinal fluid and plasma norepinephrine but not arginine vasopressin in humans

Previous studies have demonstrated alpha 2-inhibitory regulation of central nervous system (CNS) noradrenergic and arginine vasopressinergic systems. We tested the hypothesis that alpha 2-inhibition of CNS noradrenergic and vasopressinergic systems is tonic in nature by measuring the response of cerebrospinal fluid (CSF) norepinephrine (NE) and arginine vasopressin (AVP) to the alpha 2-antagonist yohimbine in 7 young normal male human subjects. We also evaluated the tonic nature of alpha 2-inhibition of the sympathetic nervous system (SNS) and of AVP release into plasma by measuring the response of plasma NE and plasma AVP to yohimbine. CSF NE was significantly higher following yohimbine as compared to placebo. In contrast CSF AVP did not differ between yohimbine and placebo conditions. Similarly, plasma NE was significantly higher following yohimbine as compared to placebo, while plasma AVP was unchanged. These results support a tonic alpha 2-inhibitory regulatory mechanism for both CNS noradrenergic systems and sympathetic outflow. Such tonic alpha 2-inhibition could not be demonstrated for regulation of AVP levels in CSF or plasma in humans.     

A comparison of the mechanisms of alpha-adrenergic inhibition of thyrotropin-stimulated adenosine 3',5'-monophosphate in cat, rat, mouse, hamster, beef, and pig tissues with the stimulatory effect of epinephrine on beef thyroid iodination: evidence for multiple, species-specific adrenergic mechanisms

Epinephrine was shown to inhibit TSH-stimulated cAMP formation in cat and pig thyroid slices and isolated rat and hamster thyroid lobes. In contrast, no such inhibitory action could be demonstrated in sheep or beef thyroid slices or mouse thyroid-trachea preparations. The inhibitory effect of epinephrine on TSH-stimulated cAMP formation in pig thyroid slices was blocked by 10 microM yohimbine, but not by 10 microM prazosin, suggestive of mediation through an alpha 2-catecholamine receptor mechanism. In cat thyroid slices, the inhibitory effect of epinephrine was blocked by both yohimbine and prazosin, suggestive of a mixed alpha-adrenoceptor mechanism. Meclofenamate and indomethacin attenuated the epinephrine response in cat, but not pig, thyroid slices, but other prostaglandin inhibitors were ineffective. Beef thyroid slices responded to epinephrine with an increase in iodide organification that is mediated through an alpha-adrenergic mechanism not blocked by propranolol. This stimulatory effect of epinephrine was abolished by both 10 microM prazosin and 10 microM yohimbine. In contrast to the inhibitory effect of the catecholamines on TSH-stimulated cAMP formation in cats, the stimulatory response of beef iodide organification to epinephrine was not modified by meclofenamate, indomethacin, or verapamil. These findings suggest that the receptor mechanisms mediating the inhibitory effect of catecholamines on cat thyroid and the stimulatory effect of catecholamines in beef thyroid slices may well be mediated by separate and as yet undefined receptor mechanisms. In contrast, the inhibitory effect of epinephrine on TSH-stimulated cAMP formation in pigs is most likely mediated through an alpha 2-adrenoceptor mechanism. These findings further document the multiplicity of catecholamine actions on thyroid function as well as the diversity observed among various species.     

Role of Alpha-2 Adrenoceptors in Regulation of Giant Migrating Contractions and Defecation in Conscious Dogs

The aim was to investigate the effects of α₂-adrenoceptor antagonsist yohimbine on colonic motility and defecation. The effects of yohimbine (0.5, 1.0, and 3.0 mg/kg) on colonic motility and defecation were studied in neurally intact dogs (N=6), dogs with extrinsic denervation of the ileocolon (N=4), and dogs with enterically isolated ileocolnic loops (N=5) equipped with strain gauge force transducers on the ileocolon. The effects of yohimbine on colonic motility and defecation were also studied in the presence of various antagonists (atropine, hexamethonium, ondansetron, FK224, and naloxone). Yohimbine evoked giant migrating contractions and defecation in a dose-independent manner in neurally intact dogs. These stimulatory effects of yohimbine were abolished by atropine and hexamethonium. In dogs with extrinsic denervation, yohimbine induced giant migrating contractions in the colon but did not stimulate defecation. In dogs with ileocolonic loops, yohimbine induced colonic motor complexes but not giant migrating contractions in the enterically isolated colon. These results indicate that α₂-adrenoceptors in the peripheral nervous system regulate giant migrating contractions by controlling the release of acetylcholine, while those in the central nervous system must be important in the regulation of defecation. This study was presented at 19th International Symposium on Neurogastroenterology and Motility in Barcelona, Spain, in October 2003. This study was supported by a Grant-in-aid from Japan Society for the Promotion of Science (NO. 13671277).     

Effects of third ventricle injection of norepinephrine analogue on LH secretion of the non-laying SIJI goose

The effect of norepinephrine analogue (NA) and norepinephrine antagonists on luteinizing hormone releasing hormone (LHRH) secretion in geese was investigated by monitoring serum levels of LH. Twenty non-laying geese were selected and randomly assigned to 4 groups of 5 each. The geese received a single intraventricular injection of saline (control), NA (12 microg/6 microl), phentolamine (1 microg/6l) plus NA or yohimbine (10 microg/6 microl). Phentolamine, a non-selective adrenoreceptor blocker was given 5 min before NA, but yohimbine, an alpha2-adrenoreceptor blocker, was delivered without NA injection. The results showed that serum levels of LH began to increase 20 min after injection of NA, and that high serum levels of LH were maintained until 105 min, followed by a decline to the basal level 125 min later. Significantly high levels of serum LH were observed at 50 and 105 min after the injection as compared with the respective control. Treatment with phentolamine completely blocked the stimulatory effect of NA on serum levels of LH in NA treated geese. Serum levels of LH increased by yohimbine treatment 5 min after the injection and the high levels of serum LH were maintained until 125 min later. Five out of 8 points were significantly high as compared with their respective control values. The present study also suggested that there is presynaptic modulation in the a-noradrenergic neural pathways which mediate the release of LHRH, and that this modulation may be carried out by alpha2-adrenoreceptor blocker.     

Effects of alpha 1-, alpha 2- and beta-adrenoceptor blockers on insulin secretion in the rat

The effects of alpha- and beta-adrenoceptor blockade on plasma concentrations of insulin and glucose were studied in the anaesthetized rat. Infusion of the alpha 1-adrenoceptor blocker prazocin (80 micrograms/min), the alpha 2-adrenoceptor blocker yohimbine (15 micrograms/min) or the non-selective alpha-adrenoceptor blocker phentolamine (15 micrograms/min) during 50 min increased plasma insulin levels by about 1.5-2.5 ng/ml. The effects of phentolamine and prazosin on circulating insulin persisted throughout the infusion whereas the effect of yohimbine seemed to be more transient. Plasma glucose levels increased slightly during infusion of prazosin, but tended to decrease in response to phentolamine and yohimbine. The beta-adrenoceptor blocker propranolol (15 micrograms/min) lowered basal plasma insulin and glucose levels. It also depressed plasma insulin during infusion of all three alpha-adrenoceptor blockers without any appreciable influence on plasma glucose. It is suggested that both alpha 1- and alpha 2-adrenoceptor as well as beta-adrenoceptors are involved in the regulation of basal insulin secretion in the rat.