Pain, wheal and flare in human forearm skin induced by bradykinin and 5-hydroxytryptamine

Pain was induced in 19 healthy individuals by double-blind injections into the forearm skin of 0.05 ml of physiological saline with or without active substances added. Bradykinin (0.5 nmol), 5-hydroxytryptamine (0.5 nmol) and a mixture of the two substances in half dosage (0.25 nmol + 0.25 nmol) caused significantly more pain than saline (p<0.05). The three test solutions also induced wheal and flare responses significantly more pronounced than saline. Bradykinin induced significantly more pain and more wheal than 5-hydroxytryptamine (p<0.05) but a significantly smaller flare (p<0.01). A dissociation between induced pain and flare was thus demonstrated.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca²⁺ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

Monoamine oxidase inhibitors and the depletion of 5-hydroxytryptamine from enterochromaffin cells by reserpine

Summary The influence of Nialamide (a monoamine oxidase inhibitor) on the depletion of 5-hydroxytryptamine from enterochromaffin cells, has been studied in rabbits. Experimental animals were given varying doses of Nialamide in N/10 HCl by single or repeated injections, followed by injections of reserpine. Control animals were given N/10 HCl followed by reserpine or by reserpine solvent. Nialamide did not prevent the depletion of enterochromaffin cell granules by reserpine in any of the experimental animals.     

5-Hydroxytryptamine2 and 5-hydroxytryptamine3 receptors mediate serotonin-induced short-circuit current in pig jejunum

The purpose of this study was to determine the effect of methysergide, ketanserin, granisetron, cisapride, and renzapride on serotonin 5-hydroxytryptamine-evoked short-circuit current in muscle and myenteric plexus-stripped pig jejunum using the Ussing chamber technique. Ketanserin, granisetron, cisapride, and renzapride all reduced the 5-hydroxytryptamine-induced increase in short-circuit current by about 50%. Combination of ketanserin and granisetron only reduced the 5-hydroxytryptamine-induced peak increase in short-circuit current by 25%. Cisapride caused a small concentration-dependent increase in short-circuit current. Atropine and hexamethonium both almost completely suppressed the cisapride-induced peak increase in short-circuit current. Ketanserin, granisetron, methysergide, and renzapride did not alter the basal short-circuit current. These results suggest that 5-hydroxytryptamine elicits an increase in short-circuit current by activating epithelial and submucosal 5-hydroxytryptamine₂ and 5-hydroxytryptamine₃ receptor subtypes. Furthermore, the short-circuit current-increasing effect of cisapride, is due to activation of at least muscarinic and nicotinic receptors.Abbreviations 5-HT 5-hydroxytryptamine, serotonin - AUC area under the curve - EC enterochromaffin - ENS enteric nervous system - GI gastrointestinal - MW molecular weight - 5-HTP-DP N-acetyl-5-hydroxytryptophyl-5-hydroxytrytophan amide - SSC short-circuit current - TTX tetrodotoxin     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.     

5-hydroxytryptamine synthesis in HL-1 cells and neonatal rat cardiocytes

Some reports showed that serotonergic system might have existed and that 5-hydroxytryptamine (5-HT) was detected in the hamster heart. The source of 5-HT in the heart, however, remains to be fully elucidated. So the present study was designed to define serotonergic system and to clarify which cell could produce 5-HT in the heart. As a result, 5-HT was detected in homogenates of HL-1 cardiomyocytes by high performance liquid chromatography with fluorescence detection, but not in those of neonatal rat non-cardiomyocytes (NMCs). And TPH and AADC mRNAs were expressed in HL-1 cardiomyocytes and neonatal rat cardiomyocytes (MCs), not in NMCs. mRNAs of 5-HT(2A) receptor were detected in both MCs and NMCs, and those of 5-HT(2B) receptor in NMCs. These findings definitively demonstrate that 5-HT is secreted from the myocytes of the heart and strongly implied that 5-HT might play a certain role in cardiac physiology.     

5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK

The purpose of this study was to elucidate the mechanism of 5-hydroxytryptamine (5-HT, serotonin) action on migration of vascular smooth muscle cells. Migration of cultured human aortic smooth muscle cells (HASMCs), evaluated using time-lapse microscopy, was significantly enhanced by 5-HT at concentrations of 1-100 nM. The enhancing effect of 5-HT on cell migration was markedly inhibited in the presence of ketanserin, a 5-HT2 receptor antagonist, but not by GR 55562, a 5-HT1 receptor antagonist. Activities of RhoA and ERK were increased by 5-HT, and the increase in cell migration by 5-HT was abolished in the presence of U0126, a MEK1/2 inhibitor, or Y-27632, a Rho-kinase inhibitor. Activation of ERK was strongly inhibited by Y-27632. 5-HT-induced formation of stress fiber and detachment of uropod (trailing edge) were abolished by Y-27632. Thus, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation by 5-HT2 receptor stimulation followed by activation of the Rho-kinase and ERK pathways.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

Deamination of 5-Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain

Abstract: K _m and V _max values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l -deprenyl. K _m values of 178 and 1170μ m , and V _max values of 0.73 and 0.09 nmol·mg protein⁻¹·min⁻¹ towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The K ₁ for 5-hydroxytryptamine as a competitive inhibitor of β-phenethylamine oxidation by MAO-B was found to be 1400 μm. The significance of these findings is discussed.     

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of l-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing l-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC–mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and ¹H and ¹³C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and l-cysteine in foods, and might act as a mutagen.     

The changes in the energy metabolism of human muscle induced by training

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.     

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats

Sarpogrelate, a specific 5-HT_2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT_2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.     

Electron-microscopic immunolabelling of vasoactive substances in human umbilical endothelial cells and their actions in early and late pregnancy

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of feto-placental blood flow. The pharmacological effects of 5-hydroxytryptamine, histamine and endothelin were examined in umbilical arteries and veins from legal terminations (gestational age 8–17 weeks, n=12) and normal term vaginal deliveries (gestational age 38–41, n=12). Immunocytochemistry of human unbilical vessels indicated that 5-hydroxytryptamine, histamine and endothelin were localised in subpopulations of endothelial cells of both artery and vein in late, but not early, pregnancy. 5-Hydroxytryptamine (10 nM–30 μM) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy. Histamine (0.1 μM–30 mM) also caused sustained contractions in all vessels from late pregnancy but only 27% of arteries and 41% of veins from early pregnancy responded. Endothelin (10 pM–30 nM) caused slow long-lasting contractions in all vessels from early and late pregnancy. Atrial natriuretic peptide and neuropeptide Y did not alter vascular tone. The endothelium may thus play an autocrine/paracrine role, by synthesizing and releasing the above reactive substances in late pregnancy to influence feto-placental blood flow. Received: 23 May 1995 / Accepted: 13 October 1995     

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.