Prejunctional Level Research Articles

Nitric oxide modulates sympathetic neurotransmission at the prejunctional level.

In spite of accumulating evidence for a modulation of sympathetic neurotransmission by endogenously produced nitric oxide (NO), it remains unclear in which parts of the vascular system and at what level this interaction takes place. The aim of the present study was to investigate the distribution of endothelial and neuronal NO synthase (NOS) along the vascular tree of the heart at the light and electron microscopic level using NADPH-diaphorase (NADPH-d) staining as a marker for NOS. In addition, the functional effects of exogenous NO on coronary vascular resistance and cardiac adrenergic nerves was studied using the isolated perfused rat heart as a model. The intraaxonal catecholamine content of adrenergic nerve fibers was visualised and morphometrically assessed by applying glyoxylic acid-induced histofluorescence. The expression of endothelial NOS in the heart was found to depend on the diameter of the blood vessel. Arteries > 100 microns always showed intense staining, whereas staining in smaller arteries and veins was considerably weaker. Smooth-muscle free vessels were essentially devoid of NADPH-d activity. In atrial and ventricular myocardium, neuronal NOS localised in autonomic nerve fibers along the entire vascular tree. Ultrastructurally, NADPH-d staining revealed adjacent localisation of NOS-positive and -negative axons, suggesting and interaxonal modulation of adjacent autonomic nerve fibers by NO. In isolated perfused rat hearts, the intracoronary application of 10(-8) M NO produced a marked decrease of coronary perfusion pressure, which was accompanied by a distinct increase in intraaxonal catecholamine levels of intramural adrenergic nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery

1 The effects of two 8-substituted analogues of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were compared with those of forskolin and isoprenaline on [3H]-noradrenaline release and vasoconstriction induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery, in the absence and in the presence of protein kinase inhibitors. 2 8-Bromo-adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP, 10-300 microM), 8-(4-chlorophenyl-thio)-adenosine 3':5' cyclic monophosphate (8-pCPT-cyclic AMP, 3-300 microM), forskolin (0.3-10 microM) and isoprenaline (1 nM-1 microM) all concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. The effect of cyclic AMP analogues was larger (2.5 fold at 300 microM) than those of cyclic AMP elevating drugs (1.6 fold at 10 microM for forskolin and 1.5 fold at 30 nM for isoprenaline). 3 At concentrations active at the prejunctional level, the four drugs had differential effects on stimulation-induced vasoconstriction, which was enhanced by the two cyclic AMP analogues, decreased by forskolin and not significantly altered by isoprenaline. 4 The [3H]-noradrenaline release-enhancing effects of 8-bromo-cyclic AMP, forskolin and isoprenaline were significantly decreased by the cyclic AMP-dependent protein kinase (PKA) inhibitor (N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5- isoquinolinesulphonamide, di-hydrochloride) (H-89; 100 nM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor, 8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 microM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor,8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 MicroM).At the same concentrations the PKA inhibitor attenuated only the nerve-induced vasoconstrictor responses obtained in the presence of 8-bromo-cyclic AMP, whereas the PKG inhibitor did not modify that obtained in the presence of 8-bromo-cycic AMP or forskolin.5. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (1 MicroM) enhanced nerve-evoked [3H]-noradrenaline release, and this effect was decreased by the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)-maleimide (GF 109203X; 100 nM). However, the latter drug did not modify the enhancing effect of 8-bromo-cyclic AMP on [3H]-noradrenaline release.6. It is concluded that activation of cyclic AMP-dependent protein kinase is involved in the enhancing effect of cyclic AMP-elevating compounds on prejunctional release of noradrenaline. In addition the results provide no clear-cut evidence for a vasodilator role of PKA.

Modulation by the endothelium of sympathetic vasoconstriction in an in vitro preparation of the rat tail artery

1. The influence of the endothelium on transmural electrical stimulation was investigated in isolated and perfused segments of the rat tail artery. Noradrenaline release (NA, quantified by h.p.l.c.-electrochemical detection) and changes in perfusion pressure (PP, measured at constant flow rate) were simultaneously recorded in unstimulated and stimulated arterial segments, in the absence and in the presence of drugs. The ratio PP/NA release (mmHg pg-1) was taken as an index of the noradrenergic effectiveness. 2. Removal of the endothelium produced an increase in NA release and PP, in unstimulated and stimulated arteries. This can be taken as evidence of an endothelium-derived inhibitory factor (EDIF) acting at the prejunctional level, inhibiting NA release. Furthermore, in unstimulated arteries, the ratio PP/NA release decreased suggesting the existence of an endothelium-derived contracting factor (EDCF). 3. Perfusion of arteries with N omega-nitro-L-arginine methyl ester (L-NAME, 10 microM) or methylene blue (MeB, 0.5 microM) had no effect on PP or NA release in unstimulated arteries. In stimulated arteries, both drugs potentiated the increase in PP without changing NA release and therefore, led to an increase in noradrenergic effectiveness. After removal of the endothelium, neither L-NAME nor MeB affected the increases in PP and NA release following electrical stimulation. 4. Carbachol (1 microM) attenuated both NA release and the increase in PP during electrical stimulation, and increased the ratio PP/NA release. L-NAME and MeB did not modify the inhibitory effect of carbachol on NA release, or the facilitatory effect of carbachol on the noradrenergic effectiveness. 5. Angiotensin II (All, 0.1 MicroM) potentiated the increase in PP, without modifying NA release following electrical stimulation, and facilitated the vasoconstriction induced by perfusion of NA. In the absence of endothelium, All potentiated both the increase in PP and NA release in arteries stimulated electrically but had no effect on the vasoconstriction induced by perfusion of NA. This suggests an endothelium dependent activity of All in this preparation.6. These findings suggest that, in the rat tail artery, sympathetic vasoconstriction is modulated by three endothelial factors: (1) nitric oxide (NO), the release ot which seems NA-dependent; (2) EDCF,predominant in the unstimulated state, the release of which; can be stimulated by All; and (3) EDIF,unmasked by removal of the endothelial layer, the release of which can be stimulated by All.

Inhibition of cholinergic neurotransmission in isolated guinea-pig main bronchi by SR 48968

SR 48968 (10 −6 to 10 −5 M) inhibited the cholinergic response of the isolated guinea-pig main brochus to electrical field stimulation. Since this effect was reversed by naloxone 10 −5 M and since SR 48968 had no effect on the contractile response to exogenous acetylcholine, we conclude that SR 48968 acts at a prejunctional level and that opioid receptors are involved. This effect was observed at concentrations approximately 75 000 times higher than those needed for blockade of tachykinin NK 2 receptors.

Norepinephrine release in rat vas deferens: Effect of rauwolscine and BHT 920

Norepinephrine (NE) overflow from field-stimulated rat vas deferens preparations was quantified directly by electrochemical detection using high performance liquid chromatography. The effect of agonist (BHT 920) and antagonist (rauwolscine) of prejunctional α2- adrenoceptors on NE overflow was assessed and compared with their effect on the smooth muscle mechanical response to field stimulation. Increasing the stimulation frequency from 2 to 30 Hz resulted in an increase in muscle tension together with an increase in NE overflow. Addition of 1 μM rauwolscine to the medium resulted in a significant increase in muscle contraction to field stimulation which reached a maximum at 5 Hz. On the other hand, NE overflow increased linearly with the frequency of stimulation within the range studied. Addition of 0.1 μM BHT 920 to the medium significantly decreased the amplitude of contractions at lower stimulation frequencies (2 to 10 Hz) but elicited no significant changes at high frequencies. BHT 920 did not significantly affect NE overflow for all range of stimulation frequency. The simultaneous recording of field-stimulation induced contractions and NE overflow indicates that in the rat vas deferens, rauwolscine acts like a pure α2 adrenoceptor antagonist at a prejunctional level. BHT 920 did not appear to affect selectively prejunctional α2 adrenoceptors but also may activate postjunctional α1 adrenoceptors.

Effects of nebivolol stereoisomers on the action of adrenaline on blood pressure, heart rate and blood levels of noradrenaline and DOPEG.

1. Nebivolol (a new beta 1-adrenoceptor blocking drug) has particular effects on the cardiovascular system, i.e. it induces hypotension without affecting cardiac function or increasing peripheral vascular resistances. In this study, we aimed to evaluate the effects of nebivolol and its stereoisomers on the actions of adrenaline (AD) at the cardiovascular level as well as at the prejunctional level (as ascertained by modification of noradrenaline (NA) and dihydroxyphenylglycol (DOPEG) plasma levels in the anaesthetized dog. 2. AD infusion (0.1 micrograms kg-1 min-1) did not induce statistically significant changes in mean blood pressure and heart rate; it caused a pronounced and sustained rise of AD levels, no significant alterations in NA levels and a marked, progressive and sustained increase in DOPEG levels. 3. Mean blood pressure was not affected by any of the nebivolol isomers. d- and dl-nebivolol in the two doses used (0.3 and 0.03 mg kg-1 in 15 min) caused a significant and dose-dependent decrease in heart rate. Plasma levels of AD and NA were not changed by any of the nebivolol isomers tested. However, all of them significantly reduced the increase in plasma levels of DOPEG induced by adrenaline infusion. 4. We conclude (1) that AD infusion in the dog facilitates NA release and that DOPEG is a good index of this effect; and (2) nebivolol appears to act at the prejunctional level, reducing the increase in NA release induced by adrenaline, as shown by the effect on DOPEG plasma levels.

M3-Muscarinic Receptor Mediates Prejunctional Inhibition of Noradrenaline Release and the Relaxation in Cat Femoral Artery

The aim of the present study was to analyse the muscarinic receptors involved in the vasodilation elicited by acetylcholine (ACh) and the carbachol inhibition of electrically-evoked [3H]noradrenaline (NA) release in cat femoral artery. For this purpose, the following receptor antagonists were used, atropine, pirenzepine (M1-antagonist), AF-DX 116 (M2-antagonist) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3-antagonist). The order of potency (pA2 values) of these drugs at postjunctional level was: atropine (9.7) greater than or equal to 4-DAMP (9.6) greater than pirenzepine (7.2) greater than AF-DX 116 (6.0), and at prejunctional level (pIC50 values) was: 4-DAMP (9.3) greater than atropine (8.5) greater than AF-DX 116 (7.1) greater than pirenzepine (5.9). These findings indicate that the muscarinic receptors mediating the vasodilation induced by ACh and the carbachol inhibition of NA release are of the M3-subtype.

Age-dependent modulation of neuropeptide y on adrenergic transmission of guinea pig vas deferens

1. 1. The effect of neuropeptide (NPY) on [ 3H]noradrenaline ([ 3H]NA) release- and on contractions evoked by field electrical stimulation (FES) was studied in vitro in vas deferens from mature and immature guinea pigs. 2. 2. The evoked tritium overflow (which reflected [ 3H]NA release) was determined by liquid scintillation spectrometry. 3. 3. Field electrical stimulation of 5 Hz (trains of 50 pulses in 20 sec intervals) evoked guanethidinesensitive contractions. 4. 4. NPY (0.01–1 μM) dose-dependently inhibited the evoked contractions in both groups of animals. NPY, 1 μM, almost completely inhibited the evoked contractions in mature animals, while those in immature guinea pigs were inhibited but only by 80.4 ± 3.6%. 5. 5. The amount of tritium overflow evoked by 5 Hz stimulation (300 pulses: 15 trains of 20 pulses in 20 sec intervals) was higher in immature guinea pigs (0.46 ± 0.03%) compared with the amount of the evoked tritium overflow in mature guinea pigs (0.39 ± 0.02%). 6. 6. NPY, 1 μM, inhibited the evoked tritium overflow. The NPY inhibition was more pronounced in vas deferens of mature (45.3 ± 2.0%) than in immature (25.1 ± 3.5%) guinea pigs. 7. 7. The results suggest that NPY modulation of adrenergic transmission at the prejunctional level increases with the maturity.

Prejunctional dopamine receptors modulate twitch responses to parasympathetic nerve stimulation in the rabbit isolated rectococcygeus muscle.

Field stimulation of the parasympathetic nerves supplying the rabbit isolated rectococcygeus muscle produced individual twitch responses that were inhibited by dopamine (1 x 10(-8)-1 x 10(-5)M). The twitch-inhibitory effect of dopamine was reversed by haloperidol or sulpiride (1 x 10(-8)-1 x 10(-5)M for either) but not by phentolamine or yohimbine (up to 1 x 10(-4)M). Haloperidol and sulpiride were potent, specific, competitive antagonists of the twitch-inhibitory effect of dopamine; their pA2 values were 8.39 and 7.75, respectively. In contrast, cis alpha-flupenthixol, fluphenazine, bulbocapnine and thioridazine were weak or inactive against dopamine. Concentrations of dopamine that inhibited the twitch response to parasympathetic nerve stimulation had little or no effect on contractions elicited by carbachol or by direct muscle stimulation after abolition of neuronal conduction by tetrodotoxin. Thus, the effects of dopamine on responses elicited by parasympathetic nerve stimulation seem to be exerted at a prejunctional level rather than directly on the smooth muscle. The twitch-inhibitory effect of dopamine was mimicked by epinine, N,N-diethyldopamine, N,N-di-n-propyldopamine, 5,6-ADTN, N,N-di-n-propyl 5,6-ADTN, 6,7-ADTN, apomorphine and Sandoz 27-403. Sulpiride reversed the effects of all these agonists. N,N-di-n-propyl-6,7-ADTN and SK & F 82526 also inhibited the twitch response but their effects were not reversed by sulpiride. SK & F 38393 and DPI had little effect on the twitch response. The pharmacological characteristics of the presynaptic dopamine receptors in the rabbit rectococcygeus muscle show that they resemble those in the cat heart and rabbit ear artery in some respects but there are differences that suggest that the presynaptic dopamine receptors in the rabbit rectococcygeus muscle constitute a specific subgroup of receptors.

Effect of prostaglandin E 2 on vascular responses of the rabbit kidney to nerve stimulation and noradrenaline, in vitro and in situ

The effect of prostaglandin E 2 on vascular responses of the rabbit kidney to renal nerve stimulation and noradrenaline was examined in vitro and in situ as a test of the hypthesis that prostaglandins of the E series may be involved in the regulation of adrenergic neuroeffector transmission. Intraarterial administration of prostaglandin E 2 to the in vitro kidney caused marked inhibition of vascular responses to nerve stimulation whereas the responses to noradrenaline were not significantly altered. In the in situ preparation, vascular responses to both nerve stimulation and noradrenaline were inhibited by prostaglandin E 2 infusion, although its effect on responses to nerve stimulation was approximately twice that observed on responses to noradrenaline. It is concluded that prostaglandin E 2 acts primarily at a prejunctional level of adrenergic neuroeffector transmission in the kidney, although a postjunctional effect has also been observed.