P. C. Chang

In vivo characterization of vasodilating muscarinic-receptor subtypes in humans.

The role of muscarinic (M)-receptor subtypes in the regulation of vascular tone has not yet been defined in humans. To analyze the role of M-receptor subtypes in the forearm resistance vasculature of normotensive volunteers (n = 20), we infused acetylcholine (ACh) and methacholine (MCh) in the presence of saline and the antagonists atropine (nonselective), pirenzepine (M1 selective), and AF-DX 116 (M2 selective), using automated R-wave-triggered venous occlusion plethysmography. Schild analysis was applied by calculating plasma concentrations of the infused compounds and determining EC50 values. ACh and MCh both caused dose-dependent vasodilation, with EC50 values of 537 and 52 nmol/L, respectively. The apparent 10-fold higher potency of MCh compared with ACh may be explained by rapid degradation of ACh in plasma. The concentration-response curve of MCh was shifted to the right by atropine, pirenzepine, and AF-DX 116, with apparent pA2 values of 8.03 +/- 0.03, 6.71 +/- 0.08, and 5.32 +/- 0.05, respectively, and slopes not different from unity. The present technique enabled us to perform M-receptor characterization by Schild analysis in humans. The affinity constants and rank order of potency--atropine > pirenzepine > AF-DX 116-suggest that cholinergic vasodilation in this vascular bed is predominantly mediated by the M3-receptor subtype. The EC50 value of MCh and the pA2 values of pirenzepine and AF-DX 116 are comparable to values reported for in vitro experiments.

Regional extraction of circulating norepinephrine, DOPA, and dihydroxyphenylglycol in humans

Dihydroxyphenylglycol (DHPG) is the main intraneuronal metabolite of the sympathetic neurotransmitter, norepinephrine (NE), and dihydroxyphenylalanine (DOPA) the immediate product of the rate-limiting step in catecholamine biosynthesis. Simultaneous measurements of regional rates of appearance (spillovers) of NE, DOPA, and DHPG in plasma have the potential to provide unique information about aspects of sympathoneural function but have not actually been measured in humans. In the present study, spillovers of DHPG, DOPA, and NE in the heart, head, leg, and lungs, were estimated from regional extraction fractions of infused [3H]-1-NE, DHPG, and [13C6]DOPA or unlabelled DOPA in humans during cardiac catheterization. There was little cardiac extraction of DHPG (7 +/- SEM 2%) or DOPA (8 +/- 4%) but substantial extraction of NE (69 +/- 4%). Values for cardiac spillover of DHPG and DOPA therefore were similar to values for the arteriovenous increment times plasma flow (arteriovenous production rate), whereas the cardiac spillover of NE averaged about 7-times the NE arteriovenous production rate. Cardiac DHPG spillover (28 +/- 3 ng/min) exceeded the spillovers of NE (9 +/- 2 ng/min) and DOPA (15 +/- 4 ng/min). In contrast, cranial DOPA spillover (159 ng/min) exceeded those of NE and DHPG by 8- and 2-fold and accounted for about 1/10 of the total spillover of DOPA into arterial plasma. In the femoral vascular bed, arteriovenous production rates of NE and DHPG were unrelated to femoral spillovers of NE and DHPG. Arterial and regional clearances of [13C6]DOPA were similar to those of unlabelled DOPA. The results suggest that (1) endogenous NE, DOPA, and DHPG all are released into the bloodstream by the heart, head, and limbs of humans; (2) DHPG and DOPA are not co-released with NE; (3) cardiac arteriovenous production rates of DOPA and DHPG can be used to indicate cardiac spillover of these catechols, whereas the cardiac NE arteriovenous production rate substantially underestimates cardiac NE spillover; and (4) estimates of limb spillover of NE and DHPG require concurrent measurements of the corresponding regional clearances.

Serotonin-induced vasodilatation in the human forearm is mediated by the "nitric oxide-pathway": no evidence for involvement of the 5-HT3-receptor

The “nitric oxide (NO)-pathway” is presumed to be involved in acetylcholine (ACh)- and serotonin (5-hydroxytryptamine, 5-HT)-mediated vasodilatation. In addition, both the 5-HT-induced transient and persistent vasodilator responses in the forearm vascular bed are abolished by the 5-HT3/5-HT4-receptor antagonist ICS 205–930 ([1H]-indol-3-carbonic-acid-tropine-ester HCl, tropisetron). We studied 5-HT-mediated vasodilatation in the forearm vascular bed of normotensive volunteers, using venous occlusion plethysmography. Intraarterial (i.a.) infusions of 5-HT, ACh, and sodium nitroprusside (SNP) all caused an increase in forearm blood flow (FBF). Single infusions of ondansetron and granisetron also caused an increase in FBF. Infusion of the NO scavenger and guanylatecyclase antagonist methylene blue (MB) did not change FBF, whereas the arginine analogue NG-monomethyl-L-arginine (L-NMMA) caused a decrease in FBF, which became less pronounced when infusions were repeated. Unlike ICS 205–930, concomitant infusions of the selective 5-HT3-receptor antagonists ondansetron (OND) and granisetron (GRAN) did not antagonize the transient or persistent vasodilator responses to 5-HT. These findings suggest the involvement of a 5-HT4-receptor. L-NMMA and MB both reduced the persistent vasodilator response to 5-HT, indicating involvement of the NO pathway. Neither MB nor L-NMMA influenced the endothelium-independent vasodilator response to SNP. ACh-induced vasodilatation was markedly potentiated by MB but was not affected by L-NMMA. The mechanism by which MB enhances the vasodilator response to ACh remains unclear.

Regional vascular effects of serotonin and ketanserin in young, healthy subjects.

The local hemodynamic effects of serotonin (5-hydroxytryptamine; 5-HT) and the selective 5-HT2 antagonist ketanserin were investigated in the forearm of 20 healthy volunteers. Single doses of 5-HT (0.1-80 ng/kg/min) and ketanserin (5-125 ng/kg/min) were administered intra-arterially. The relative alpha 1-adrenergic receptor and 5-HT2 blocking potencies of ketanserin were investigated using intra-arterial infusions of cumulative doses of methoxamine (0.1, 0.3, and 0.5 microgram/kg/min), tyramine (0.25, 0.50, and 1.25 microgram/kg/min), and 5-HT (10, 30, and 80 ng/kg/min) together with a low dose (5 ng/kg/min) and a high dose (50 ng/kg/min) of ketanserin. Forearm blood flow was measured by venous occlusion plethysmography. Heart rate and intra-arterial blood pressure were recorded semicontinuously. Intra-arterial infusion of 5-HT induced an initial transient vasodilatation, followed by a steady vasodilatation for the low doses of 5-HT (0.1-10 ng/kg/min; p less than 0.05). A steady vasoconstriction was only obtained at the highest dose of 5-HT. Ketanserin induced a dose-dependent increase in forearm blood flow from 15 ng/kg/min (p less than 0.05) onward. The vasodilatation induced by 5-HT (1 ng/kg/min) was significantly enhanced by ketanserin (125 ng/kg/min; p less than 0.05), whereas the vasoconstriction elicited by 5-HT (80 ng/kg/min) was reversed by ketanserin (50 ng/kg/min; p less than 0.05), thus confirming that 5-HT2 receptors were stimulated by 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

No functional involvement of 5-hydroxytryptamine1A receptors in nitric oxide-dependent dilatation caused by serotonin in the human forearm vascular bed.

Summary The vascular effects of serotonin (5-hydroxytryptamine, 5-HT) are complex and heterogeneous. In human forearm, we showed that low doses of 5-HT cause marked but transient vasodilatation followed by a persistent vasodilator response. In in vitro and in animal experiments, 5-HT induced release of nitric oxide (NO) through stimulation of endothelial 5-HT,-like receptors. In the present study, we investigated involvement of the “NO pathway” and possible involvement of the 5-HT1A receptor subtype in 5-HT-induced persistent vasodilator response. In 8 healthy volunteers, we infused 5-HT (0.1, 0.3, and 1 ng/kg/min) and the selective 5-HT1A receptor agonist flesinoxan (15, 45, and 150 ng/kg/min) intraarterially (i.a.) with NG-monomethyl-L-arginine (L-NMMA 30 (μ/kg/min) or saline. Forearm blood flow (FBF) was measured by automated R-wave-triggered venous occlusion plethysmography. Forearm vascular resistance (FVR) was derived from simultaneously recorded i.a. blood pressure (BP) and FBF. 5-HT dose-dependently decreased FVR (p < 0.001). The persistent vasodilator response to 5-HT appears to be mediated by NO release, as suggested by its complete abolition by L-NMMA (p < 0.001). Flesinoxan decreased FVR slightly, but only at high doses (p < 0.05). The present findings indicate that 5-HT1A receptors are not functionally involved in 5-HT-mediated vasodilatation in human forearm.

Assessment of Peripheral Vascular Effects of Antimigraine Drugs in Humans

The vascular beds of the forearm and finger can be used to study the peripheral effects of antimigraine drugs under normal and pathologic circumstances. We have investigated the novel antimigraine drug sumatriptan, a selective agonist for 5HT1 receptors. Its antimigraine effect may be attributed, at least in part, to constriction of cranial arteriovenous anastomoses (AVAs). In assessing the peripheral vascular effects of sumatriptan we used a forearm and finger blood flow model. Forearm blood flow (FBF) is mainly determined by resistance vessels, whereas finger blood flow (FiBF) mainly involves skin vessels, which contain many AVAs. Changes in FBF and FiBF can be assessed using venous occlusion plethysmography. Changes in AVA flow are determined by measuring the patency of the vascular beds of the forearm and hand to well-defined radiolabeled microspheres, which are injected into the brachial artery. We report the effects of sumatriptan on FBF, FiBF and AVA flow when administered into the brachial artery of healthy volunteers, and discuss the peripheral vascular effects of therapeutic doses of sumatriptan when given subcutaneously in migraine patients during and between attacks.

Arterial and venous effects of serotonin in the forearm of healthy subjects are not age-related

The influence of age on the regional arterial and venous effects of serotonin (5-HT) was investigated in 13 young (aged 22–31 years) and seven older (aged 50–69 years) healthy volunteers. Single-dose infusions of 5-HT (1, 10, and 80 ng/kg/min) and of the 5-HT2 receptoi antagonist ritanserin (50, 150, and 500 ng/kg/min) were administered into the brachial artery. Subsequently, the relative arterial and venous effects of the highest dose of 5-HT were investigated. Forearm blood flow (FBF) and maximum venous outflow (MVO) were measured by venous occlusion plethysmography. Heart rate (HR) and intraarterial (i.a.) blood pressure were recorded semicon tinuously. In both age groups, 5-HT induced an initia transient arterial dilation, followed by a persistent increase in FBF for the doses of 1 and 10 ng/kg/min and a relative small decrease in FBF for the highest dose. of both age groups, the highest dose of 5-HT induced a similar large reduction in MVO (p < 0.05 for both). The reduction in MVO was attenuated by ritanserin (500 ng/kg/ min, p < 0.05 for both groups). In the younger subjects, this dose of ritanserin also unmasked an arterial dilator effect of the highest dose of 5-HT (p < 0.05). The single infusions of ritanserin did not influence FBF significantly in either study group. No significant differences were observed between the age groups. These results show that in the forearm of healthy subjects arterial and venous vascular responses to 5-HT were not age-related. In the arterial vascular bed, 5-HT acted predominantly as a vasodilator; at high doses, mainly venous vasoconstriction was observed. That ritanserin did not significantly influence FBF in either age group provides evidence that 5-HT2 receptors are not involved in maintenance of basal vascular tone in the vascular bed investigated.

Effects of 5-hydroxytryptamine on Capillary and Arteriovenous Anastomotic Blood Flow in the Human Hand and Forearm and in the Pig Hind Leg

The effects of intraarterially infused serotonin (5-HT) on capillary and arteriovenous anastomotic (AVA) blood flow were investigated in the hand and forearm of 19 healthy volunteers, and in the hind leg of six anesthetized pigs using radioactive microspheres with a diameter of 15 μm. The 5-HT2-receptor antagonist ketanserin was used in an attempt to identify the receptors involved. None of the drugs in the doses used induced systemic hemodynamic effects. Low doses of 5-HT significantly increased forearm blood flow with a maximum response at the dose of 1 ng/kg/min (68 ± 14%, p < 0.05), whereas only at the highest dose of 80ng/kg/min was a net decrease in forearm blood flow measured ( - 28 ± 6%. p < 0.05). Conversely, finger blood flow was not influenced by the lower doses of 5-HT, whereas a major reduction was observed at the highest dose ( - 90 ± 3%). Ketanserin increased both total forearm blood flow and AVA blood flow. The drug blunted the constrictor response to 5-HT in the forearm but only slightly attenuated this response in the finger. The percentage AVA blood flow in the human hand and forearm was not influenced by an infusion of 5-HT at 80 ng/kg/min alone. However, after pretreatment with ketanserin, which itself increased the AVA component, this dose of 5-HT significantly reduced AVA flow. In the pig, total femoral blood flow was not influenced by 5-HT, but AVA blood flow was significantly reduced and capillary skin blood flow increased. It is concluded that in both humans and pigs intraarterially infused 5-HT decreases AVA blood flow but that this effect is seemingly not mediated by 5-HT2 receptors but, as previously reported in the porcine carotid circulation, may involve 5-HT1-like receptors. The fact that low doses of 5-HT did not induce a net vasodilation in the finger is attributed to the relative absence of skeletal muscles and abundance of AVAs in the finger.

Potentiation of the pressor effect of oral and intravenous tyramine during administration of the selective MAO-A inhibitor moclobemide in healthy volunteers

The interaction between tyramine and the new short-acting and reversible mono amine oxidase inhibitor moclobemide was investigated in a double-blind placebo-controlled study in six healthy volunteers. There were two consecutive study periods of 8 days during which the subjects received moclobemide three x 200 mg daily or placebo. On day 5 of each study period changes in systolic blood pressure (SBP) were determined after incremental intravenous bolus doses of tyramine and on days 6, 7 and 8 changes in SBP were determined after oral tyramine (100, 200 and 300 mg, respectively). Oral tyramine was administered together with a standard breakfast, before which moclobemide had been given. On days 5- 8 blood was taken for determination of blood drug levels. On days 6-8 blood samples were taken before and at 15, 30 and 45 min after tyramine administration for determination of plasma tyramine and plasma norepinephrine concentrations. When SBP had increased by approximately 30 mmHg no further doses of either intravenous or oral tyramine were given. Moclobemide was well tolerated by all subjects. Plasma trough levels of moclobemide were within the therapeutic range. The tyramine induced increases in SBP were greater during moclobemide than during placebo. After intravenous tyramine the dose-response curve for SBP was shifted to the right by a factor of approximately 3. When compared to placebo the pressor response to 100 mg tyramine orally was not significantly different, but the pressor response to the other two doses was enhanced during moclobemide.

Raised plasma renin and prorenin in rheumatoid vasculitis.

The value of plasma renin and its inactive precursor, prorenin, were examined as a marker for vasculitis in rheumatoid arthritis (RA). Plasma renin and prorenin rise when the renin-angiotensin system is activated; an isolated increase of prorenin may be a marker for microvascular complications in diabetes mellitus. Renin concentrations in plasma obtained from 34 patients with RA (seven with vasculitis, 27 controls) were measured under standard conditions, before and five days after stopping non-steroidal anti-inflammatory drugs; creatinine clearance was also measured. At first the median renin concentration in the patients with vasculitis was 19 (range 12-63) mU/l (normal less than 61 mU/l) and in the controls 9 (3-43) mU/l. The median prorenin concentration in patients with vasculitis was 233 (144-428) mU/l (normal less than 358 mU/l) and in the controls 144 (25-364) mU/l. Renin and prorenin concentrations increased significantly in both groups after withdrawal of nonsteroidal anti-inflammatory drugs. The creatinine clearance was similar in both groups and did not correlate with renin concentrations. In conclusion, it was found that, unlike patients with diabetes mellitus, patients with RA with vasculitis had slightly raised concentrations of both renin and prorenin. These findings signal activation of the renin-angiotensin system and might indicate early cardiac or renal involvement by vasculitis.