Nils Grundström

Prejunctional alpha2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission

Abstract Ring preparations obtained from the guinea pig trachea contracted on short trains of electrical field stimulation. These contractions were mediated by activation of cholinergic nerves since they were abolished by atropine or tetrodotoxin. In the presence of beta blocking drugs noradrenaline and adrenaline dose-dependently inhibited contractions induced by field stimulation. By contrast, contractions on exogenous acetylcholine were left completely unaffected. It is concluded that the adrenergic agonists inhibited cholinergic neurotransmission by a prejunctional action. In order to characterize the noradrenaline receptor the effects of alpha 1 and alpha 2 blockers were evaluated using the Schild plot. For comparison experiments were also conducted on the guinea pig aorta and electrically stimulated guinea pig ileum. The results indicate that in guinea pig trachea and ileum noradrenaline inhibits cholinergic neurotransmission by acting on prejunctional alpha 2 receptors whereas in guinea pig aorta it induces contraction by stimulating alpha 1 receptors.

Suppression by neuropeptide Y of capsaicin-sensitive sensory nerve-mediated contraction in guinea-pig airways

1 In the present study we have examined whether neuropeptide Y (NPY) interferes with non‐adrenergic, non‐cholinergic nerve‐mediated contractions and relaxations in the guinea‐pig airways. In these experiments we have used ring preparations of bronchi and trachea, incubated in the presence of atropine, propranolol and indomethacin (each 1 μm). 2 The contractile response to electrical stimulation of non‐adrenergic, non‐cholinergic nerve fibres was suppressed by NPY and NPY 13–36 in a concentration‐dependent manner, these agents having similar inhibitory potencies. NPY caused a more complete inhibition than the C‐terminal fragment. 3 NPY affected neither the basal tension nor the substance P‐evoked contraction in the bronchi and trachea and did not interfere with nerve‐mediated, non‐adrenergic relaxation in the trachea. 4 On the basis of these results, it is suggested that NPY may act on the terminals of sensory neurones in the airways to prevent antidromic, excitatory neurotransmission by inhibiting transmitter release.

Cloning and expression of a fish α2-adrenoceptor

1 Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α2‐adrenoceptor pharmacology. We now report the cloning of the α2‐F, a fish skin α2‐receptor from the cuckoo wrasse (Labrus ossifagus). 2 Degenerate oligonucleotides corresponding to conserved regions of the human α2‐adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human α2‐adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3 A clone (pTB17BS) consisting of ∼5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and ∼2 kb of 5′‐untranslated sequence. The deduced amino acid sequence of this protein showed 47–57% identity with the human α2‐adrenoceptors and thus appeared to encode a fish α2‐adrenoceptor. 4 In the 5′‐untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α2‐F might be regulated by cyclic AMP, calcium and/or steroids. 5 The α2‐F was expressed in COS‐7 cells and radioligand binding studies were performed with [3H]‐rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 ± 0.1 nm and a Bmax of 5.7 ± 1.0 pmol mg−1 of protein. 6 Competition curves for the displacement of specific [3H]‐rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine> p‐aminoclonidine > B‐HT 920 > (−)‐noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results show that α2‐F has characteristics of both the human α2‐C10 and α2‐C4 and that it might represent an ancestral α2‐adrenoceptor subtype.

Role of nitric oxide and cyclic GMP as mediators of endothelium-independent neurogenic relaxation in bovine mesenteric artery.

Electrical field stimulation (EFS) of phenylephrine-contracted bovine mesenteric arteries pretreated with guanethidine elicited a relaxation that amounted to roughly 40%. This relaxation was sensitive to tetrodotoxin pretreatment, suggesting a neurogenic origin. The EFS-induced relaxation was correlated to an increase in cGMP level, from 14.2 +/- 2.5 pmol/g wet wt in nonstimulated arteries to 31.6 +/- 3.4 pmol/g wet wt after 1 minute of EFS. cAMP values were not affected by EFS. Methylene blue (5 microM) and the compound LY 83583 (10 microM), inhibitors of soluble guanylate cyclase, inhibited the EFS-induced relaxation by 60% and 50%, respectively. Zaprinast (1 microM), a selective inhibitor of cGMP degradation, significantly (p = 0.005) potentiated the EFS-induced relaxation. The relaxation induced by EFS in bovine mesenteric arteries exhibits characteristics similar to the relaxations evoked by organic nitroesters and endothelium-dependent vasodilators, both of which are suggested to be mediated by cGMP and probably with nitric oxide as the common activator of the cGMP system. The possible involvement of nitric oxide as a mediator of EFS-induced relaxations was investigated with the use of known modulators of endogenous nitric oxide production. Preincubation of the arteries with 1 mM arginine or 1 mM N-alpha-benzoyl-L-arginine, both reported to potentiate endogenous nitric oxide production, or 5 mM L-canavanine, 0.25 mM NG-monomethyl-L-arginine, or 0.1 mM NG-nitro-L-arginine, alleged inhibitors of endogenous nitric oxide production, were without effect on the relaxation induced by EFS. However, pyrogallol, a generator of superoxide anions, was a potent inhibitor of relaxations induced by EFS in bovine mesenteric arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of pigment aggregating α2‐adrenoceptors of fish melanophores by use of different agonists after partial irreversible receptor inactivation

1 The affinity for, and the intrinsic efficacy on, postsynaptic melanosome aggregating α2‐adrenoceptors of fish melanophores was studied for B‐HT 920, clonidine, medetomidine, noradrenaline, phenylephrine and UK‐14,304. Investigations were carried out by evaluating the effects of progressive, irreversible inactivation of the α2‐adrenoceptors by benextramine. 2 The double reciprocal plots of equieffective concentrations of B‐HT 920, clonidine, noradrenaline and phenylephrine were linear, which indicated that these compounds exerted their effects, mainly, through interaction with one receptor site. 3 The affinity for the α2‐adrenoceptor selective agonist B‐HT 920, was found to be about 1000 times higher than the affinity for the α1‐adrenoceptor selective agonist phenylephrine. 4 The corresponding plot of equieffective concentrations of medetomidine was not linear, which may indicate that this imidazole compound exerted its effect through more than one receptor site. However, when phenoxybenzamine was used in place of the more selective benextramine, a linear relationship was obtained.

Comparative studies on nerve- and noradrenaline-induced melanosome aggregation within different species of fish.

1. The aggregation of melanosomes within melanophores of the cuckoo wrasse (Labrus ossifagus; belonging to the family Labridae) has, on pharmacological grounds, been shown to be mediated by postsynaptic alpha 2-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. 2. In the present paper we have investigated some American species belonging to the Labridae, Haemulidae, Embiotocidae, Clinidae and Pleuronectidae. 3. In all instances, except in the case of sargo (Haemulidae), we could demonstrate that melanosome aggregation probably was mediated by postsynaptic alpha 2-adrenoceptors which mediate their effect by inhibiting the adenylate cyclase of the melanophores. 4. Although these receptors apparently, on pharmacological grounds, may be classified as alpha 2-adrenoceptors it was also concluded that there is a phylogenetic divergence among these receptors.

MCH-induced pigment aggregation in teleost melanophores is associated with a cAMP reduction.

It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.

A novel neurogenic vasodilator mechanism in bovine mesenteric artery.

The presence of a neurogenic vasodilator mechanism was investigated in isolated bovine mesenteric arteries (BMAs) that were precontracted with phenylephrine. Electrical field stimulation induced tetrodotoxin-sensitive relaxations in guanethidine-pretreated BMAs. The relaxation occurred after a delay of about 5-8 seconds and amounted to 25-35% in different sets of experiments. The relaxation was not affected by classical receptor antagonists such as atropine (1 μM), cimetidine (3.9 μM), clemastine (2.8 μM), naloxone (1.2 μM), 8- phenyltheophylline (1 μM), propranolol (3.4 μM), ritanserin (5 μM), or droperidol (13 μM). The nicotinic acetylcholine-receptor stimulant l,l-dimethyl-4-phenyl-piperazinium iodide (10 μM) was without effect on the relaxation, and removal of the endothelium of the arteries also had no effect. The bee venom component apamin (1 μM), which has been shown to block the nonadrenergic, noncholinergic relaxation in intestinal and vascular smooth muscle from other species, was also found to be without effect on the relaxation induced by electrical field stimulation in BMAs. Pretreatment of the arteries with capsaicin (1 μM) had no effect per se and did not affect the relaxation induced by a subsequent stimulation. Capsaicin has been suggested to release neurotransmitter and eventually deplete neurons containing substance P and calcitonin gene-related peptide. Furthermore, exogenously applied calcitonin gene-related peptide (1-100 nM), substance P (10 nM-1 μM), and vasoactive intestinal peptide (0.3-30 nM) gave relaxations amounting to less than 10%. It is postulated that electrical field stimulation induces a neurogenic relaxation of a nonadrenergic, noncholinergic nature in BMAs. The relaxation is not dependent on an intact endothelium and seems not to be mediated by any of the known vasodilatory neuropeptides.

Comparison of heart rate measured by Polar RS400 and ECG, validity and repeatability

Abstract Aims: The purpose of this study was to investigate criterion-related validity and test–retest repeatability of the heart rate monitor Polar RS400 versus electrocardiogram (ECG). Methodology: Ten healthy subjects, 19–34 years, performed a cycle ergometer test 5 min on each load (50, 100 and 150 W). Heart rate (HR) was measured with ECG and Polar RS400 and recorded digitally. After at least one hour resting the test was repeated. Major findings: The results showed a significant correlation between HR measured by ECG and by Polar RS400 with correlation coefficients ranging from 0.97 to 1.00. In test 1 the mean difference ± 2SD between HR Polar and HR ECG was 0.7 ± 4.3 bpm and in test 2, 0.2 ± 3.2 bpm. In the repeated tests, the mean difference of HR between test 2 and test 1 ± 2SD was 3.2 ± 11.9 bpm with ECG and 2.6 ± 14.3 bpm with Polar RS400 and these differences were not statistically significant. Conclusion: This study indicates good criterion-related validity and test–retest repeatability of Polar RS400. Differences observed at individual levels should be noticed, but are not considered to be clinically important. Polar RS400 is thus well suited for recording HR during physical activity and exercise training.

Melanophores in isolated scales of Labrus berggylta (Ascanius): innervation and alpha2-adrenoceptor-mediated pigment aggregation

Abstract 1. Innervation and adrenoceptor-mediated pigment granule (i.e. melanosome) aggregation were investigated in melanophores of isolated scales from Labrus berggylta . 2. Tetrodotoxin (10 −6 M) and guanethidine (10 −4 M) blocked the electrically induced melanosome aggregation. 3. Electrically induced melanosome aggregation was inhibited by yohimbine (10 −6 M) but not by prazosin (10 −6 M). 4. The adrenoceptor-agonist order of potency was: medetomidine (alpha 2 -adrenoceptor-selective) > noradrenaline ≥ B-HT 920 > clonidine > dopamine > isoprenaline. 5. Forskolin (10 −5 M) blocked the medetomidine-induced melanosome aggregation. 6. There were no signs of beta-adrenoceptor-mediated melanosome dispersion. 7. These results indicate that the melanophores are innervated by adrenergic nerve fibres, and the aggregation of the melansomes is mediated by an alpha 2 -adrenoceptor, which probably acts via inhibition of adenylate cyclase.