Bo Fallgren

Co-existence of neuropeptides and differential inhibition of vasodilator responses by neuropeptide Y in guinea pig uterine arteries.

The co-localization of substance P (SP) with calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) with neuropeptide Y (NPY) of the guinea pig uterine artery were investigated with immunocytochemistry. The SP/CGRP fibre population was distinct from the VIP/NPY fibre population. Both types of fibres ran in the medial-adventitial border, and appeared as coarsed and fine varicosed. Uterine arterial dilatation was evoked by acetylcholine (ACh), SP, CGRP, and VIP in precontracted arteries as examined by a sensitive in vitro method. Strong relaxations were seen by ACh, CGRP and VIP. NPY had no relaxant effect per se but was found to be a potent inhibitor of vasodilation induced by ACh and SP, while relaxations induced by VIP and CGRP were unaffected. The functional significance of NPY in the uterine artery may to a large extent be to increase tension not only by potentiation of contraction but additionally by inhibiting vasodilator responses.

Mechanisms of Action of Endothelin on Isolated Feline Cerebral Arteries: In vitro Pharmacology and Electrophysiology

Vascular endothelium has been found to produce a strong and potent vasoconstrictor peptide, endothelin. In this study, we have examined basic mechanisms underlying the contractile response of cerebral vessels to endothelin using in vitro pharmacology and electrophysiology. It was found that endothelin produced strong concentration-dependent contractions of circular segments of the feline middle cerebral artery. The response was slow in onset and long lasting. The vessels showed a remarkably strong tachyphylactic reaction upon repeated exposure to endothelin. The contractile effect of endothelin was not modified by the α-adrenoceptor antagonist phen-tolamine (10−6 M) or the 5-hydroxytryptamine antagonist ketanserin (10−6 M). Mechanical removal of the endothelium decreased potassium contractions while the maximum response to endothelin was only slightly reduced. There was no change in sensitivity of the cerebral artery to endothelin. The addition of a calcium antagonist (10−6 M diltiazem or 3 × 10−8 M nimodipine) or removal of extracellular calcium from the buffer solution did not change the sensitivity of the artery to endothelin but the maximum response to endothelin was reduced by between 40 and 60% by these procedures. The resting membrane potential of the cat middle cerebral artery was –62.8 ± 3.5 mV. There was no significant depolarization in conjunction with cumulative administration of endothelin in concentrations below 1 × 10−9 M. However, bursts of excitatory junction potentials were occasionally seen in response to high concentrations of endothelin (5 × 10−9 M). The findings suggest that the contractile response to endothelin of cat cerebral arteries involved influx of extracellular calcium through voltage-sensitive calcium channels and is in part mediated via a voltage-insensitive mechanism. Further work is necessary to define the intracellular actions of endothelin.

Magnesium deficiency in coronary artery disease and cardiac arrhythmias

Magnesium deficiency may be of importance for the pathogenesis and the clinical outcome of several cardiac disorders, including coronary artery disease and cardiac arrhythmias 11-71. Magnesium deficiency is frequently found [8-lo], and the administration of magnesium has proved to be of therapeutic value [S, 8. 9. 11-16] in subjects with cardiac diseases. Magnesium is the fourth most plentiful cation in the body [4]. Intracellularly. the concentration of magnesium is only exceeded by that of potassium. The level of free intracellular magnesium is, however, low and varies with time, conditions, and subcellular structure [17]. The myocardial content of magnesium is comparatively high : the concentration in the ventricles is somewhat higher than in the atria [4, 18-20]. It has been shown that the ability of cardiac muscle (and vascular smooth muscle) to retain magnesium when placed in a magnesium deficient solution is high compared with other types of muscle tissue [4, 7, 211. The myocardium is nevertheless vulnerable to magnesium deficiency, i.e. heart muscle cell degeneration, fibrosis, necrosis and calcifications may be seen in conjunction with magnesium depletion [4, 22, 231. In heart tissue magnesium stimulates oxidative phosphorylation. Na-K-ATPase. adenylatecyclase, and myofibrillar contraction [ 17, 241. Furthermore, the complex of MgATP2+ is the substrate of a number of enzymatic reactions, including those noted above [ 1 71. A vasodilator action of magnesium was first observed in 1932 by Hazard & Wurmser [251. Three years later Zwillinger [ 31 reported anti-arrhythmic properties of magnesium. Since then evidence has accumulated indicating that magnesium is a strong relaxant of most types of blood vessel, including coronary arteries [2. 6 , 7, 26-28]. and that magnesium can convert cardiac tachyarrhythmias [ 5,

Inhibition of neuropeptide Y-induced potentiation of noradrenaline-induced vasoconstriction by PP56 (d-myo-inositol 1,2,6-tris-phosphate)

1 Although neuropeptide Y (NPY) is a potent vasoconstrictor in many vascular beds, nanomolar concentrations of this peptide potentiate the noradrenaline‐induced contractions in rabbit gastroepiploic and femoral arteries, and guinea‐pig mesenteric and uterine arteries. 2 The potentiating effect of NPY on noradrenaline‐induced contraction was present in endothelium‐denuded femoral arteries. 3 The potentiating effect of NPY on noradrenaline‐induced contraction was antagonized by PP56 (d‐myo‐inositol 1,2,6‐trisphosphate) in low concentrations (down to 0.1 nm). This antagonistic effect was observed in all four types of vessels studied. Contractions induced by noradrenaline, histamine, endothelin‐1 and potassium were not altered by PP56 in concentrations upto 1 μm in femoral artery of rabbit. 4 We provide evidence that a non‐peptide (PP56) can selectively antagonize NPY‐induced effects in rabbit and guinea‐pig peripheral arteries without affecting the vasoconstrictor response to noradrenaline.

Characterization of adrenoceptor mechanisms in isolated guinea-pig uterine arteries

The adrenoceptors of the guinea-pig uterine artery were characterized pharmacologically. Circular segments of the artery, approximately 2 mm long, and with an external diameter of 250 micron, were mounted in miniaturized tissue baths. Noradrenaline, methoxamine and phenylephrine (concentrations ranging from 10 nM to 1 mM), in the presence of propranolol (0.1 microM) and cocaine (1 microM), induced concentration-dependent contractions of the arterial segments. Clonidine (10 nM to 0.1 mM) was less effective in producing contraction of the vessel. Prazosin (10 nM to 1 microM) antagonized noradrenaline-induced contractions; its pA2 was 7.68. Rauwolscine (10 nM to 1 microM) had no effect on noradrenaline-induced contractions. Isoprenaline (10 nM to 0.1 mM) in the presence of prazosin (1 microM) and cocaine (1 microM) had no relaxant effect on arteries contracted submaximally by prostaglandin F2 alpha (5 microM). Cocaine or normetanephrine treatment did not influence the noradrenaline-induced contractions. It is concluded that in guinea-pig uterine arteries, amine-induced contractions are mediated predominantly by alpha 1-adrenoceptors and that in this arterial preparation, relaxant beta-adrenoceptor effects and neuronal or extraneuronal uptake are of minor if any importance.

Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction by an intracellular calcium-dependent mechanism

The potentiating effect of neuropeptide Y (NPY) was examined by testing the influence of putative inhibitors of calcium entry on the NPY-enhanced contractile response to noradrenaline in the guinea pig uterine artery. In order to examine the involvement of voltage sensitive calcium entry mechanisms we recorded the effect of noradrenaline and NPY on the membrane potential. NPY (100-300 nM) enhanced noradrenaline-evoked vasoconstriction. The potentiation by NPY was most prominent in low noradrenaline concentrations (30-300 nM) and the pD10 (-log molar concentration of agonist eliciting 10% of maximum contraction) value was increased from 6.43 +/- 0.07 to 6.97 +/- 0.11 (P < 0.001, n = 6). Inhibition of extracellular calcium influx shifted concentration-dependently to the right the concentration-response curve for noradrenaline but potentiation by NPY still remained. The intracellular calcium chelator quin-2 AM selectively abolished the NPY-induced enhancement of the contractile response to noradrenaline. In contrast, quin-2 AM (10-30 microM) had no inhibitory effect on the contractile response to noradrenaline per se. It is suggested that NPY initiates an intracellular calcium-sensitive mechanism which increase alpha-adrenoceptor sensitivity. This results in a significant increase of sarcoplasmic calcium and stronger contractile responses to noradrenaline.

Neuropeptide Y in cerebrovascular function: comparison of membrane potential changes and vasomotor responses evoked by NPY and other vasoconstrictors in the guinea pig basilar artery

The membrane depolarization and vasomotor response evoked by NPY and other vasoconstrictors were compared in guinea pig basilar artery. Concentrations below the pD2 value of amines and PGF2 alpha induced contractions without significant membrane depolarization, while higher agonist concentrations depolarized the membrane slightly. Potassium-induced contractions were paralleled by strong depolarization. NPY evoked a slow depolarization which correlated to vasoconstriction over a wide concentration range. The mechanism of activation did not appear to involve the endothelium. The results suggest that NPY induces prolonged cerebrovascular smooth muscle tone by evoking longlasting depolarization, at least partly in conjunction with activation of voltage-operated calcium channels.

Characterization of specific binding sites for α-trinositol (D-myo-inositol 1,2,6-trisphosphate) in rat tissues

Alfa-trinositol (or D-myo-inositol 1,2,6-trisphosphate) was recently found to, e.g., inhibit agonist-induced vasoconstriction and display antiinflammatory properties. However, its mechanism of action is unknown, although effects on Ca2+ fluxes, perhaps by interfering with endogenous inositol phosphate(s), have been suggested. Here we describe the existence of specific [3H]alpha-trinositol binding sites and compare these with binding sites for naturally occurring inositol phosphates. For this purpose we developed a tritiated analog of alpha-trinositol and used it in a centrifugation binding assay on extensively washed membranes from rat tissues. The degree of specific [3H] alpha-trinositol binding was markedly increased as a result of the many wash steps, indicating the existence of endogenous binding inhibitor(s). A single population of [3H] alpha-trinositol binding sites, displaying a KD of 159 nM and a Bmax of 71 pmol/mg protein, was present in cardiac membranes assayed at pH 7.4. Similar binding site densities were detected also in liver > lung > brain. The relative density of [3H] alpha-trinositol sites in cardiac membranes was 8-fold higher than [3H]Ins(1,4,5)P3 but 2-fold and 4-fold lower than [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding sites, respectively. Competition binding studies indicated the ability of Ins(1,3,4,5)P4 and InsP6, but not Ins(1,4,5)P3, to potently displace [3H] alpha-trinositol binding. Conversely, unlabelled alpha-trinositol showed relatively low potency vs. [3H]InsP6, but the novel inositol phosphate was virtually equipotent with Ins(1,3,4,5)P4 in inhibiting [3H]Ins(1,3,4,5)P4 binding. Finally, analyses of binding at different pH and ionic conditions revealed differences between alpha-trinositol and the three other previously studied inositol phosphates, although distinct similarities between alpha-trinositol and Ins(1,3,4,5)P4 were again observed.

Involvement of perivascular neuropeptide Y nerve fibres in uterine arterial vasoconstriction in conjunction with pregnancy.

The perivascular neuropeptide Y (NPY) innervation and its relation to adrenergic nerves of uterine arteries from non-pregnant and pregnant guinea pigs was analyzed immunocytochemically. The NPY content of the uterine artery was, in addition, measured radioimmunologically (RIA). Vasomotor effects of NPY per se and in combination with other vasoconstrictors were examined using a sensitive in vitro method. Pregnancy did not visibly affect density and distribution of NPY-immunoreactive fibres. The NPY fibres contained in addition immunoreactivity to dopamine-beta-hydroxylase (marker for noradrenergic neurons). RIA revealed a slight decrease of NPY content during pregnancy, probably due to the increased smooth muscle volume of uterine arteries. The contractile effect of NPY on uterine arteries was weak, while vasoconstriction induced by various agonists was potentiated by NPY, particularly during pregnancy. It is concluded that perivascular NPY-containing nerve fibres may be involved in the dramatic blood flow alterations that occur in the uterine circulation in connection with pregnancy and partus.

The effect of α-trinositol (d-myo-Inositol 1,2,6-trisphosphate) on formalin-evoked spinal amino acid and prostaglandin E2 levels

The effect of the inositol trisphosphate analog alpha-trinositol on noxious-evoked behavior, amino acid and prostaglandin E2 (PGE2) release was examined in unanesthetized rats using intrathecal microdialysis probes. Subcutaneous injection of 50 microliters 5% formalin solution produced two phases of pain-like behavior and significant elevation of glutamate, aspartate, glycine, taurine and serine during phase 1. PGE2 concentrations were increased during both phases 1 and 2. Intraperitoneal delivery of 300 mg/kg alpha-trinositol significantly suppressed both phases 1 and 2 of formalin-induced behavior and the associated elevation of amino acids and PGE2. These data demonstrate that the antinociceptive effect of alpha-trinositol corresponds to suppression of noxious-evoked release of amino acids and PGE2 from the spinal cord.