Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C.

1. The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release-enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically-induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre-incubated with [3H]-noradrenaline. 2. 8-Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S-I noradrenaline release through cyclic AMP-dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 microM) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release-enhancing effect of McNeil A 343 (10 microM) on noradrenaline release was also not altered by the 5-lipoxygenase inhibitor, BW A4C. 3. The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin-dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin. 4. It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are sensitive to N-ethylmaleimide, but not pertussis toxin.

1. The identity of the G-proteins involved in prejunctional alpha 2-adrenoceptor signal transduction in mouse atria was examined by use of the G-protein inactivators N-ethylmaleimide and pertussis toxin. 2. The alpha 2-adrenoceptor partial agonist clonidine (0.03 microM) inhibited the electrical stimulation-induced (S-I) outflow of radioactivity from mouse atria which were incubated with [3H]-noradrenaline and stimulated at 5 Hz. The partial alpha 2-adrenoceptor agonist St 363 (10 microM) inhibited the S-I outflow of radioactivity at the lower stimulation frequency of 2.5 Hz. The inhibitory effects of these compounds were not altered in mice pretreated with pertussis toxin (1.5 micrograms, i.v.). 3. The alpha 2-adrenoceptor antagonist, idazoxan (0.1 microM), increased the S-I outflow of radioactivity from mouse atria stimulated at 5 Hz, and this effect was not altered in atria from mice pretreated with pertussis toxin. 4. The inhibitory effects of clonidine and St 363 and the facilitatory effect of idazoxan on the S-I outflow of radioactivity from mouse atria were significantly less in atria incubated with N-ethylmaleimide (NEM, 3 microM) for 60 min before the [3H]-noradrenaline incubation. 5. The results suggest that prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are NEM-sensitive, but pertussis toxin insensitive.     

Pertussis toxin differentiates between alpha 1- and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release from rat kidney cortex

In slices of rat kidney cortex incubated in [3H]noradrenaline, the alpha 1-adrenoceptor agonist methoxamine (10 microM), the alpha 2-adrenoceptor agonist clonidine (0.1 microM), as well as adenosine (10 microM), inhibited the electrical stimulation-induced (S-I) outflow of radioactivity, at a stimulation frequency of 1 Hz. Prior treatment of rats with pertussis toxin (25 micrograms/kg i.v.), which abolished the negative inotropic effect of carbachol (10 microM) on isolated atria, prevented the inhibition caused by methoxamine, but not that caused by clonidine or adenosine. At a stimulation frequency of 5 Hz, the alpha 2-adrenoceptor antagonist idazoxan (0.1 microM) and the prostaglandin synthesis inhibitor indomethacin (10 microM) both facilitated the S-I outflow of radioactivity, and neither of these effects were altered by pertussis toxin. These results suggest that a pertussis toxin sensitive G-protein is involved in alpha 1-adrenoceptor inhibition of noradrenaline release, but not in alpha 2-adrenoceptor, adenosine or prostaglandin inhibition.     

Facilitation of noradrenaline release from sympathetic nerves through activation of ACTH receptors, beta-adrenoceptors and angiotensin II receptors.

1. In rabbit pulmonary artery and left atrial strips previously incubated with [3H]-noradrenaline, the active fragment of adrenocorticotropic hormone (ACTH 1-24, 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity when a cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM) was present, but not in the absence of these drugs. In rabbit pulmonary artery a facilitatory effect of ACTH 1-24 (0.1 microM) was also observed when only cocaine (30 microM) was present. 2. ACTH 1-24 (0.1 microM) did not affect the S-I outflow of radioactivity from rat atria, rat pulmonary artery or guinea-pig pulmonary artery, either in the presence or in the absence of the cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM). These results suggest that the presence of facilitatory prejunctional ACTH receptors may be restricted to rabbit sympathetic nerve endings. 3. Angiotensin II (0.01 microM), but not isoprenaline (0.1 microM) or ACTH 1-24 (0.1 microM), significantly enhanced the S-I outflow of radioactivity from rabbit pulmonary artery. In the presence of phentolamine (1 microM) to block inhibitory alpha 2-adrenoceptors, the facilitatory effect of angiotensin II (0.01 microM) was significantly enhanced, and a significant facilitatory effect of isoprenaline (0.1 microM) and of ACTH 1-24 (0.1 microM) was then revealed. These results suggest that feedback inhibition of noradrenaline release, mediated through the prejunctional alpha 2-adrenoceptor mechanism, buffers increases in noradrenaline release during activation of facilitatory prejunctional receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for facilitatory and inhibitory muscarinic receptors on postganglionic sympathetic nerves in mouse isolated atria.

1. McNeil A 343 (10 microM-30 microM) enhanced the fractional stimulation-induced (S-I) outflow of radioactivity from mouse isolated atria which had been incubated with [3H]-noradrenaline. The enhancing effect of McNeil A 343 was not altered by hexamethonium (300 microM) suggesting that it was not due to an action at nicotinic receptors. It is also unlikely that McNeil A 343 enhanced the S-I outflow of radioactivity in mouse atria by blocking neuronal reuptake of noradrenaline since the effect persisted in the presence of cocaine (30 microM). 2. The facilitatory effect of McNeil A 343 on the S-I outflow of radioactivity was attenuated by atropine (0.3 microM), pirenzepine (0.2 microM or 1.0 microM), dicyclomine (1.0 microM) and methoctramine (1.0 microM) and was thus due to activation of muscarinic receptors. 3. In contrast to the effect of McNeil A 343, another muscarinic receptor agonist, carbachol (3.0 microM) significantly decreased the S-I outflow of radioactivity. The receptors through which McNeil A 343 acts to enhance the S-I outflow of radioactivity appear to be distinct from inhibitory prejunctional muscarinic receptors. The relatively M 1-selective antagonist, pirenzepine (0.2 microM), attenuated the facilitatory effect of McNeil A 343 whereas a higher concentration (1.0 microM) was required to block the inhibitory effect of carbachol. Conversely, the relatively M2-selective antagonist, methoctramine (0.1 microM), blocked the inhibitory effect of carbachol but a higher concentration of methoctramine (1.0 microM) was required to block the facilitatory effects of McNeil A 343.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of histamine on the resting and stimulation-induced release of [3H]-noradrenaline in guinea-pig isolated atria.

1 The sympathetic transmitter stores of guinea-pig isolated atria were labelled with [3H]-noradrenaline. The effects of histamine (0.3 to 100 mumol/l) on resting and stimulation-induced (S-I, 2 Hz for 10 s) release of radioactivity were investigated. 2 Histamine, in low concentrations (0.3 and 1 mumol/l) had no effect on resting release but inhibited S-I release of radioactivity. The inhibition was abolished by the H2-receptor antagonist, cimetidine (10 mumol/l) and also by the H1-receptor antagonist, mepyramine (1 mumol/l). 3 The inhibitory actions of histamine on S-I release were not due to indirect effects involving alpha-adrenoceptors, beta-adrenoceptors, muscarinic cholinoceptors or prostaglandin synthesis. 4 Histamine in a high concentration (100 mumol/l) increased the resting and S-I release of radioactivity. The increase in resting release was abolished by the neuronal uptake blocking drug cocaine (30 mumol/l) but the increase in S-I release was only partially blocked by cocaine.     

Alpha 2-autoreceptor subclassification in rat isolated kidney by use of short trains of electrical stimulation.

1 Rat kidneys were perfused with Krebs-Henseleit solution and incubated with [3H]-noradrenaline. The renal nerves were electrically stimulated at either 1 Hz for 30 s or 100 Hz for 0.06 s. The stimulation induced (S-I) outflow of radioactivity was taken as an index of endogenous noradrenaline release. 2 At a frequency of 1 Hz for 30 s the alpha-adrenoceptor antagonists BRL 44408 (0.01, 0.1 microM) and imiloxan (0.1, 1.0 microM) enhanced S-I outflow of radioactivity. However, at a frequency of 100 Hz for 0.06 s the alpha-adrenoceptor antagonists, idazoxan (0.1, 1.0 microM), imiloxan (0.1, 1.0 microM), BRL 44408 (0.1, 1.0 microM), BRL 41992 (0.1, 1.0 microM) and prazosin (0.01 microM) failed to enhance S-I outflow of radioactivity. 3 Thus, the rat isolated kidney stimulated at 100 Hz for 0.06 s, avoids autoinhibition by endogenous noradrenaline and alpha-adrenoceptor antagonist affinities (pKB) at the prejunctional alpha-autoreceptor were estimated without disturbance by the endogenous activator. 4 The alpha 2-adrenoceptor agonist, clonidine, inhibited the S-I outflow of radioactivity with a maximum of 90% and an EC50 of 7.2 nM. 5 All alpha-adrenoceptor antagonists used caused parallel shifts of the concentration-response curve for clonidine to the right. The rank order of potencies was: rauwolscine (alpha 2A/B) > idazoxan (alpha 2A/B) > phentolamine (alpha 2A/B) > WB 4101 (alpha 2A) > BRL 44408 (alpha 2A) > BRL 41992 (alpha 2B) > prazosin (alpha 2B) = imiloxan (alpha 2B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple prejunctional actions of angiotensin II on noradrenergic transmission in the caudal artery of the rat.

1. Angiotensin II produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The threshold concentrations of angiotensin II for enhancement of S-I efflux (between 0.03 and 0.1 microM) and of the stimulation-evoked vasoconstrictor responses (about 0.3 microM) were 10-1000 times higher than those that have been found for several other vascular preparations. 2. The AT1 angiotensin II receptor antagonist losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of S-I efflux by 1 and 3 microM angiotensin II and the enhancement of vasoconstrictor responses by 1 microM angiotensin II. Surprisingly, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced S-I efflux to a much greater extent than did 0.1 microM angiotensin II alone. Moreover, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced stimulation-evoked vasoconstrictor responses, in contrast to the lack of effect of 0.1 microM angiotensin II alone. 3. In a concentration of 0.01 microM, the angiotensin II AT2 receptor antagonist PD 123319 did not affect the enhancement of either S-I efflux or vasoconstrictor responses by angiotensin II. However, in a higher concentration (0.1 microM), PD 123319 antagonized the enhancement of both the S-I efflux and vasoconstrictor responses by angiotensin II. 4. In concentrations of 0.01 and 0.1 microM, PD 123319 prevented the marked enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses produced by the combination of 0.1 microM angiotensin II and 0.01 microM losartan. 5. The potentiation by losartan (0.01 microM) of the facilitatory effect of 0.1 microM angiotensin II on S-I efflux and on stimulation-evoked vasoconstriction was still observed in the presence of either the cyclooxygenase inhibitor indomethacin (3 microM), or the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. The findings confirm our previous suggestion that, in the rat caudal artery, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 7. The synergistic prejunctional interaction of 0.01 microM losartan and 0.1 microM angiotensin II may be due to either the unmasking by losartan of a latent population of angiotensin II receptors also subserving facilitation of transmitter noradrenaline release, or alternatively, losartan may block an inhibitory action of angiotensin II on transmitter noradrenaline release which normally opposes its facilitatory effect.     

Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria.

1. We investigated the modulation by bradykinin (BK) of electrically induced noradrenaline release in rat isolated atria preincubated with [3H]-noradrenaline. 2. BK (1-100 nM) enhanced significantly the stimulation-induced outflow of radioactivity in a concentration-dependent manner with a calculated EC50 of 0.58 nM. 3. Des-Arg9-BK (0.1-100 nM), a selective B1 receptor agonist, did not modify the stimulation-induced outflow of radioactivity. Hoe 140 (10 nM), a selective B2 receptor antagonist, but not [Leu8]-des-Arg9-BK (100 nM), a selective B1 receptor antagonist, blocked the facilitatory effect of BK. 4. The effect of BK was not affected by diclofenac (1 microM), a cyclo-oxygenase inhibitor. Bisindolylmaleimide (1 microM), a protein kinase C inhibitor, significantly reduced the facilitatory effect of BK (10 nM), angiotensin II (0.3 microM) and phorbol dibutyrate (0.1 and 1 microM) but not of fenoterol (1 microM). 5. The results suggest that BK enhances noradrenaline release via a prejunctional B2 kinin receptor in the rat atrium. The effect appears to involve protein kinase C as a second messenger.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

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).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of endogenous and synthetic prostanoids, the thromboxane A2 receptor agonist U-46619 and arachidonic acid on [3H]-noradrenaline release and vascular tone in rat isolated kidney.

1. Rat kidneys were perfused with Krebs-Henseleit solution and the perfusion pressure was monitored. After incubation with [3H]-noradrenaline the renal nerves were stimulated. The stimulation-induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. The effect of prostaglandins on perfusion pressure, pressor responses to renal nerve stimulation (RNS) and S-I outflow of radioactivity was assessed. 2. Prostaglandin E2 (PGE2, 0.06 and 0.6 microM), PGF2 alpha (0.6 microM), PGI2 (0.6 and 3 microM) and iloprost (0.6 microM) increased perfusion pressure and enhanced pressor responses to RNS. These facilitatory effects of the prostaglandins were not a result of an enhanced transmitter release. In contrast, PGE2 dose-dependently inhibited, whereas the other prostaglandins failed to modulate S-I outflow of radioactivity. PGE2 (0.6 microM) also enhanced pressor responses to exogenous noradrenaline. 3. Arachidonic acid (1 microM) increased perfusion pressure and enhanced pressor responses to RNS. These effects were abolished in the presence of indomethacin (10 microM) suggesting that local production of prostaglandins from exogenous arachidonic acid was responsible for this facilitation. However, arachidonic acid (1 microM) did not modulate S-I outflow of radioactivity. Arachidonic acid (10 microM), despite causing a marked increase in perfusion pressure, failed to alter pressor responses to RNS and only slightly inhibited S-I outflow of radioactivity. 4. The thromboxane A2 (TxA2) receptor agonist U-46619 (0.1 microM) increased vascular tone and enhanced pressor responses to RNS. These effects were blocked by the newly developed selective TxA2 receptor antagonist, daltroban (BM 13505; 3 microM), suggesting that these facilitatory effects of U-46619 were due to activation of TxA2 receptors. However, U-46619 failed to alter the S-I outflow of radioactivity from rat isolated kidney. 5. The alpha 1-adrenoceptor agonist methoxamine (1 microM) also increased perfusion pressure and enhanced pressor responses to RNS without affecting the S-I outflow of radioactivity in the presence of the prostaglandin synthesis inhibitor indomethacin (10 microM). 6. The results suggest that PGE2 modulates noradrenaline release through an inhibitory prejunctional receptor mechanism. There is no evidence for prejunctional PGF2 alpha, PGI2 or TxA2 receptors in the rat isolated kidney. All prostaglandins increased vascular tone in the rat isolated kidney and this alone may provide a condition for enhanced pressor responses to RNS since methoxamine also enhanced pressor responses to RNS without affecting S-I outflow of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prejunctional effects of cromakalim,nicorandil and pinacidil on noradrenergic transmission in rat isolated mesenteric artery

1 The present study investigated the effects of cromakalim, nicorandil and pinacidil on resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [³H]-noradrenaline. The efflux of radioactivity evoked by field stimulation of peri-arterial sympathetic nerves (pulses at 2 Hz frequency in trains of 60 s duration) was taken as an index of transmitter noradrenaline release. 2 Cromakalim (1–100 μm ) and nicroandil (1–1000 μm ) produced minor effects on resting and S-I effluxes of radioactivity, but these did not exhibit concentration-dependency. 3 Pinacidil (1–1000 μm ) produced concentration-dependent increases, in both resting and S-I effluxes of radioactivity. With 1000 μm pinacidil, resting and S-I effluxes were increased to approximately 348% and 358% of their respective control values. 4 The effects of pinacidil on resting and S-I effluxes were unaltered when the neuronal amine transport system was inhibited by desipramine (1 μm ). 5 Inhibition of monoamine oxidase with pargyline (100μm ) treatment markedly reduced the enhancement of resting efflux by 1000 μm pinacidil but did not alter its effect on S-I efflux. It is proposed that the enhanced resting efflux produced by pinacidil without pargyline treatment consists of deaminated [³H]-noradrenaline metabolites formed from [³H]-noradrenaline displaced from transmitter storage vesicles by pinacidil. 6 The enhancement of S-I efflux by pinacidil does not appear to involve disruption of α₂-adrenoceptor auto-inhibition of transmitter release since equi-effective concentrations of phentolamine (1 μm ) and pinacidil (1000 μm ) produced additive effects on S-I efflux, whereas increasing the concentration of phentolamine from 1 to 2m produced no further increases in S-I efflux. 7 In conclusion this, study has provided no evidence of a prejunctional inhibitory effect of the potassium channel openers cromakalim, nicorandil and pinacidil on transmitter noradrenaline release. However, the findings with pinacidil suggest that, in high concentrations, pinacidil displaces noradrenaline from transmitter stores, such that deaminated noradrenaline metabolites are released from the nerve terminals. Furthermore, pinacidil enhances S-I transmitter noradrenaline release, possibly by blocking neuronal potassium channels.     

Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway

Summary In mouse atria previously incubated with [³H]-noradrenaline, carbachol (1.0 μmol/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the α-adrenoceptor antagonist phentolamine (1.0 μmol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 μmol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 μmol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,100 μmol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 μmol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 μmol/l) plus IBMX (100 μmol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 μmol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the postreceptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 μg). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 μg). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein. Send offprint requests to M. Costa at the above address     

Facilitation of noradrenaline release from sympathetic nerves in rat anococcygeus muscle by activation of prejunctional beta-adrenoceptors and angiotensin receptors.

1. Isolated preparations of rat anococcygeus muscle were incubated with [3H]-noradrenaline and the efflux of radioactivity induced by stimulation of intramural sympathetic nerves was used as a measure of release of transmitter noradrenaline. Isometric contractile responses were also measured. 2. Angiotensin I (0.03 microM) and angiotensin II (0.03 microM) produced non-sustained contractile responses and enhanced the stimulation-induced (S-I) effluxes of radioactivity as well as the contractile responses to electrical stimulation. These effects were blocked by the angiotensin II receptor antagonist saralasin (0.03 microM), and the effect of angiotensin I, but not angiotensin II, was blocked by the angiotensin converting enzyme inhibitor captopril (0.1 microm). 3. The findings indicate that there are both pre- and postjunctional receptors for angiotensin II and that angiotensin I is converted to angiotensin II in the anococcygeus muscle preparation. 4. Isoprenaline (0.1 microM) slightly enhanced the S-I efflux of radioactivity, and produced a greater enhancement after neuronal uptake blockade with desipramine (0.03 microm) and alpha-adrenoceptor blockade with phentolamine (1 microM). 5. The facilitatory effect of isoprenaline on S-I efflux of radioactivity was abolished by propranolol (0.3 microM), but was not affected by low concentrations of saralasin (0.03 microM) or captopril (0.1 microM) which abolished the effect of angiotensin I. The findings suggest that isoprenaline acts directly on prejunctional beta-adrenoceptors to enhance S-I noradrenaline release, rather than indirectly by releasing angiotensin II from within the tissue. Higher concentrations of saralasin (0.1 microM) or captopril (5 microM) did block the facilitatory effect of isoprenaline. The significance of this finding is not clear.     

Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery.

1. The effects of NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), haemoglobin and methylene blue have been examined on vascular reactivity in the rat isolated caudal artery. The effects of L-NNA and sodium nitroprusside were also investigated on the stimulation-induced (S-I) efflux of noradrenaline in the rat caudal artery. 2. L-NNA (10 microM) and L-NAME (10 microM) significantly attenuated the vasodilator responses to acetylcholine (1 nM-1 microM), but had no effect on vasodilator responses to papaverine (1-100 microM). 3. Vasoconstrictor responses to sympathetic nerve stimulation (3 Hz, 10 s), noradrenaline (0.01-1 microM), methoxamine (1-10 microM), 5-hydroxytryptamine (0.01-0.3 microM), phenylephrine (0.1-10 microM), endothelin-1 (10 nM) and KCl (40 mM) were significantly enhanced by 10 microM L-NNA. L-NAME (10 microM) caused a significant enhancement of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in endothelium-intact, but not in endothelium-denuded tissues. 4. Haemoglobin and methylene blue (both 10 microM) enhanced the vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline. The enhancements were absent in endothelium-denuded arterial segments. 5. In endothelium-denuded arterial segments precontracted with phenylephrine, the vasodilator responses to the nitric oxide donor, sodium nitroprusside (0.1-300 nM) were decreased by increasing the level of precontraction. 6. L-NNA (10 microM) had no effect on the S-I efflux of radioactivity from arteries in which transmitter stores had been labelled with [3H]-noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C involvement in maintenance and modulation of noradrenaline release in the mouse brain cortex.

1. The role of protein kinase C in the modulation of noradrenaline release was investigated in mouse cortical slices which were pre-incubated with [3H]-noradrenaline. The aim was to investigate the hypothesis that protein kinase C is activated during high levels of transmitter release to maintain transmitter output. 2. The protein kinase C activators, phorbol myristate acetate (0.01-0.3 microM) and to a greater extent 4 beta-phorbol 12,13-dibutyrate (0.01-0.3 microM) significantly enhanced stimulation-induced noradrenaline release whereas 4 alpha-phorbol 12,13-dibutyrate (0.1 microM) which does not activate protein kinase C was without effect. The effect of the protein kinase C activator, phorbol myristate acetate, on noradrenaline release was attenuated by the protein kinase C inhibitor, polymyxin B (21 microM) which by itself inhibited stimulation-induced noradrenaline release. 3. Protein kinase C was down-regulated by 10 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta-phorbol 12,13-dibutyrate (0.1 microM) on noradrenaline release was abolished as was the inhibitory effect produced by polymyxin B. This indicates that polymyxin B was acting selectively at protein kinase C. 4. The inhibitory effect of polymyxin B on noradrenaline release, when expressed as a percentage of the appropriate frequency control, was constant at 1, 5 and 10 Hz. Furthermore, the ratio of release at 5 Hz to that at 10 Hz was not altered by protein kinase C down-regulation, indicating that there is no additional effect of protein kinase C at higher stimulation frequencies. 5. When transmitter release was elevated by blocking alpha 2-adrenoceptor auto-inhibition with idazoxan (0.1 microM) or K+ channels with tetraethylammonium (300 microM), the elevation in transmitter release was significantly attenuated by protein kinase C down-regulation, suggesting an involvement of protein kinase C. 6. We conclude that protein kinase C is involved in the modulation of noradrenaline release over a wide range of stimulation frequencies, in addition to a role when noradrenaline release is elevated by presynaptic mechanisms.     

Adrenaline activation of prejunctional beta-adrenoceptors in guinea-pig atria.

1. Adrenaline in a concentration of 1.0 microM depressed the stimulation-induced efflux of tritium from the guinea-pig atria incubated with [3H]-noradrenaline, whereas adrenaline in a concentration of 0.5 nM significantly enhanced the stimulation-induced efflux of tritium. This enhancement was blocked by metoprolol (0.1 microM) and thus appears to be mediated by beta-adrenoceptors. 2. In guinea-pig atria incubated with unlabelled adrenaline and then with [3H]-noradrenaline, both catecholamines were released by field stimulation. In such atria metoprolol, practolol, oxprenolol or propranolol decreased the stimulation-induced efflux of tritium. These effects did not occur if the atria were incubated with unlabelled noradrenaline and then with [3H]-noradrenaline, suggesting that neuronally released adrenaline activates prejunctional beta-adrenoceptors. 3. The effect of oxprenolol in decreasing the release of tritium from guinea-pig atria, incubated with unlabelled adrenaline and then with [3H]-noradrenaline was greater in the presence of phentolamine. This may reflect the alpha-adrenoceptor blocking activity of oxprenolol.     

Angiotensin II receptors involved in the enhancement of noradrenergic transmission in the caudal artery of the spontaneously hypertensive rat.

1. The effects of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319, on actions of angiotensin II in isolated caudal arteries of spontaneously hypertensive (SH) and age-matched normotensive (Wistar-Kyoto) rats were compared. 2. Angiotensin II (0.1-3 microM) produced concentration-dependent increases in perfusion pressure in artery preparations from both SH and Wistar-Kyoto (WKY) rats, the maximal increase in the SH rat being significantly greater than the increase in WKY rats. The increase in perfusion pressure in preparations from both strains of rats was prevented by losartan (0.1 microM) and unaffected by PD 123319 (0.1 microM), indicating that the vasoconstrictor action of angiotensin II is subserved by AT1 receptors. 3. Angiotensin II (0.1-3 microM) produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of caudal artery from both SH and WKY rats, in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux produced by angiotensin II (1 microM) was significantly greater in artery preparations from WKY rats than in preparations from SH rats, whereas the maximum enhancement of stimulation-evoked vasoconstrictor responses was greater in preparations from SH rats than in those from WKY rats. 4. In artery preparations from both WKY and SH rats, the AT1 angiotensin II receptor antagonist, losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of both S-I efflux and vasoconstrictor responses by 1 microM angiotensin II. 5. The combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced both the S-I efflux and stimulation-evoked vasoconstrictor response in caudal artery preparations from WKY rats, whereas 0.1 microM angiotensin alone was ineffective. The AT2 receptor antagonist PD 123319 (0.01 and 0.1 microM) prevented the enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses by the combination of angiotensin II and losartan. 6. In contrast to findings in WKY preparations and those previously obtained for arteries from another normotensive strain (Sprague-Dawley), in artery preparations from SH rats there was no synergistic interaction between losartan and angiotensin II. Rather, combinations of 0.1 microM angiotensin II and PD 123319 (both 0.01 and 0.1 microM) enhanced S-I [3H]-noradrenaline efflux, whereas 0.1 microM angiotensin II alone was without effect. Moreover, losartan (0.1 microM) prevented the enhancement of S-I efflux by the combination of angiotensin II and PD 123319. 7. The present findings indicate that in the caudal artery of WKY and SH rats, and as previously found in Sprague-Dawley preparations, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 8. As previously suggested for Sprague-Dawley caudal artery preparations, the synergistic prejunctional interaction of losartan and 0.1 microM angiotensin II in caudal artery preparations from WKY rats may be due to either the unmasking by losartan of a latent population of angiotensin II receptors subserving facilitation of transmitter noradrenaline release, or blockade by losartan of an inhibitory action of angiotensin II on transmitter release. 9. The synergistic interaction of PD 123319 and 0.1 microM angiotensin II in caudal arteries of SH rats may also be explained by either of the mechanisms proposed for the normotensive strains, but the involvement of different receptor subtypes would need to be postulated for each of the proposed mechanisms.