Stimulation-induced Efflux Research Articles

α 2-Adrenoceptor activation inhibits noradrenaline release in human and rabbit isolated renal arteries

The aim of the present study was to investigate α 2-adrenoceptor modulation of noradrenaline release in supervised strips of human and rabbit renal arteries. The arteries were field-stimulated after incubation with [ 3H]noradrenaline. The stimulation-induced outflow of radioactivity was taken as an index of noradrenaline release. At a high stimulation frequency (4 Hz), the α 2-adrenoceptor agonist clonidine (0.1 μmol/l) failed to inhibit stimulation-induced outflow of radioactivity in human and rabbit renal arteries whereas the α 2-adrenoceptor agonist UK 14304 (0.1 μmol/l) did inhibit stimulation-induced outflow. The inhibitory effect of UK 14304 in human renal arteries was blocked by the α 2-adrenoceptor blocking drug rauwolscine (1 μmol/l). At a lower stimulation frequency (2 Hz), both clonidine and UK 14304 inhibited stimulation-induced outflow of radioactivity from rabbit renal arteries; both effects were blocked by rauwolscine. Rauwolscine by itself enhanced stimulation-induced outflow of radioactivity in both preparations. The results suggest that activation of prejunctional α 2-adrenoceptors in human and rabbit renal arteries inhibits noradrenaline release. Neuronally released noradrenaline exerts inhibitory feed-back modulation of its own release through activation of prejunctional α 2-adrenoceptors. At a higher stimulation frequency most of the prejunctional a2-adrenoceptors are already occupied by endogenous noradrenaline and clonidine fails to inhibit noradrenaline release since it seems to act as a partial agonist at these prejunctional α 2-adrenoceptors.

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. These results tentatively ascribe facilitatory muscarinic receptors as belonging to the Ml subtype and inhibitory muscarinic receptors as belonging to the M2 subtype. 4. The non-selective muscarinic receptor antagonist, atropine, enhanced the S-I outflow of radioactivity, suggesting that there was tonic activation of inhibitory prejunctional muscarinic receptors by endogenous acetylcholine released from parasympathetic nerves. However, pirenzepine (0.03 pM-LO microM) did not decrease the S-I outflow of radioactivity, suggesting that under the conditions of the present study, facilitatory muscarinic receptors are not tonically activated by endogenous acetylcholine.

Chronic cocaine treatment does not alter rat striatal D 2 autoreceptor sensitivity to pergolide

Rat superfused striatal slices, preloaded with [ 3H]dopamine, were electrically stimulated and the stimulation-induced outflow of radioactivity was taken as an index of dopamine release. In the presence of 10 μM nomifensine, exposure of striatal slices to unlabelled dopamine (0.3 μM) for 6 min prior to stimulation, significantly reduced stimulation-induced outflow. In contrast, a 21-min exposure to dopamine did not significantly alter stimulation-induced outflow. These results suggest that D 2 receptors modulating dopamine release in the rat striatum may be rapidly desensitized in vitro. Rats were pretreated for 14 days with cocaine HCl (10 mg/kg/day i.p.) or saline. A progressive enhancement of locomotor activity in cocaine-treated rats over the pretreatment period compared to that in saline-treated rats indicated a behavioural sensitization to cocaine. The inhibitory effect of pergolide (1, 10 and 100 nM) on stimulation-induced outflow from striatal slices obtained from cocaine-pretreated rats was not different from that in slices obtained from saline-pretreated rats. Therefore no evidence was obtained for either a desensitization or a supersensitivity of striatal D 2 autoreceptors by chronic cocaine administration.

Angiotensin II generation in the rat vena cava: Stimulation of local synthesis by ?-adrenoceptor activation

There is considerable evidence to suggest that isolated tissues have the capacity to generate angiotensin II and that angiotensin II thus generated may enhance noradrenergic neurotransmission. In the present study the possibility that there may be local formation of angiotensin II within the rat vena cava and its consequence to noradrenergic transmission has been investigated. The angiotensin II precursors, angiotensin I and a synthetic tetradecapeptide renin substrate, each enhanced the stimulation-induced efflux of radioactivity from tissues previously incubated with 3H-noradrenaline. The effects of angiotensin I were blocked by the converting enzyme inhibitor captopril and the receptor antagonist saralasin, indicating local conversion to angiotensin II. The effect of the tetradecapeptide was blocked by saralasin, but not by captopril, suggesting either a direct effect of this peptide or conversion to angiotensin II by a pathway not involving angiotensin converting enzyme. The beta-adrenoceptor agonist isoprenaline enhanced noradrenergic transmission in the rat vena cava, relaxed guinea-pig trachea precontracted with carbachol and increased heart rate in rat isolated atria. Captopril and saralasin blocked the effect of isoprenaline in the vena cava, but did not alter its effects in the atria or trachea. This suggests that in the rat vena cava the facilitation of noradrenergic transmission by isoprenaline may involve stimulation of local angiotensin II production.

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

In mouse atria previously incubated with [3H]-noradrenaline, carbachol (1.0 mumo1/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the alpha-adrenoceptor antagonist phentolamine (1.0 mumol/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 mumol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 mumol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 mumol/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 mumol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 mumol/l) plus IBMX (100 mumol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 mumol/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 post-receptor 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 micrograms). 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 micrograms). 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.

Contractile effects of endothelin‐I and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K+ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-1-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca2(+)-free solution abolished the endothelin-1-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca2(+)-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to alpha-adrenoceptor blockade. Pretreatment with endothelin-1 (10(-9) M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of 3H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [125]endothelin-1 binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.

Effects of methylenedioxymethamphetamine on the release of monoamines from rat brain slices

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamine release were investigated in superfused slices of rat striatum and hippocampus. MDMA (10 μM) increased the resting release of radioactivity from slices incubated in [ 3H]dopamine, [ 3H]5-hydroxytryptamine or [ 3H]noradrenaline. These effects of MDMA (10 μM) were blocked by the neuronal uptake inhibitors, cocaine (10 μM), fluoxetine (1 μM) and desmethylhnipramine (1 μM), respectively. MDMA (10 μM) enhanced the stimulation-induced efflux of radioactivity from slices incubated in [ 3H]noradrenaline but not from slices incubated in [ 3H]5-hydroxytryptamine or [ 3H]dopamine. These results demonstrate for the first time a direct noradrenaline-releasing action of MDMA and differential effects of MDMA on the stimulation-induced release of noradrenaline, dopamine and 5-hydroxytryptamine from rat superfused brain slices.

Prostaglandin E2 inhibits noradrenaline release and purinergic pressor responses to renal nerve stimulation at 1 Hz in isolated kidneys of young spontaneously hypertensive rats

The effects of prostaglandin E2 (PGE)2 and the thromboxane A2 (TxA2) receptor agonist U-46619 on noradrenaline release and pressor responses to renal nerve stimulation (RNS) at 1 Hz were investigated in isolated kidneys of spontaneously hypertensive rats (SHR; 5-7 weeks) and age-matched Wistar-Kyoto rats (WKY). 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. Absolute S-I outflow of radioactivity was lower in SHR than in WKY but pressor responses to RNS were greater in SHR than in WKY. Tetrodotoxin (1 mumol/l) abolished S-I outflow of radioactivity and pressor responses to RNS in both strains. PGE2 (0.06 mumol/l) inhibited S-I outflow of radioactivity in SHR but not in WKY kidneys. PGE2 (0.6 mumol/l) inhibited S-I outflow of radioactivity in both strains. In SHR, PGE2 (0.6 mumol/l) decreased pressor responses to RNS, but increased them in WKY. In WKY, but not in SHR kidneys, pressor responses to RNS were markedly reduced by the alpha 1-adrenoceptor antagonist prazosin (0.1 mumol/l). The prazosin-resistant pressor responses to RNS were blocked by alpha, beta-methylene adenosine triphosphate (ATP; 1 mumol/l). In kidneys of SHR, pretreated with 6-OH-dopamine (50 mg/kg intravenously, 24 and 48 h before isolation of the kidneys) to destroy sympathetic nerve endings, pressor responses to RNS and S-I outflow of radioactivity were almost abolished. U-46619 (0.1 mumol/l) increased perfusion pressure in SHR and WKY kidneys and this effect was blocked by the TxA2 receptor antagonist daltroban (BM 13505; 3 mumol/l). U-46619 did not significantly modulate S-I outflow of radioactivity. The results suggest that activation of prejunctional PGE2 receptors in kidneys of SHR and WKY inhibits noradrenaline release. The prejunctional inhibitory PGE2 receptor mechanism on renal sympathetic nerves seems to operate more effectively in SHR than in WKY. There is no evidence for prejunctional TxA2 receptors in the kidneys of SHR or WKY. Pressor responses to RNS at 1 Hz in SHR kidneys seem to be due entirely to release of a purinergic co-transmitter from renal sympathetic nerves, and PGE2 possibly reduces pressor responses to RNS by inhibiting release of this purinergic co-transmitter.

PERTUSSIS TOXIN ATTENUATES ANGIOTENSIN II BUT NOT β‐ADRENOCEPTOR FACILITATION OF NORADRENALINE RELEASE FROM RAT KIDNEY CORTEX

1. Angiotensin II (AII; 0.01 and 0.1 mumols/L), angiotensin I (AI, 0.1 mumols/L) and the beta-adrenoceptor agonist isoprenaline (0.1 mumols/L) all facilitated the stimulation-induced outflow of radioactivity from slices of rat kidney cortex incubated in [3H]-noradrenaline. 2. Treatment of rats with pertussis toxin (25 and 50 micrograms/kg i.v.) to inactivate G-proteins attenuated the facilitation caused by AII and AI, but not that caused by isoprenaline. 3. The hypothesis that isoprenaline enhances noradrenaline release by generating AII to activate facilitatory prejunctional AII receptors is not supported by the present study. The hypothesis predicts that pertussis toxin, by inactivating the G-proteins associated with AII receptors, should have inhibited the facilitatory effect of isoprenaline. This did not occur.

Pertussis toxin differentiates between α1- and α2-adrenoceptor-mediated inhibition of noradrenaline release from rat kidney cortex

In slices of rat kidney cortex incubated in [ 3H]noradrenaline, the α 1-adrenoceptor agonist methoxamine (10 μM), the α 2-adrenoceptor agonist clonidine (0.1 μM), as well as adenosine (10 μM), 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 μg/kg i.v.) which abolished the negative inotropic effect of carbachol (10 μM) on isolated atria, prevented the inhibition caused by methoxamine, but not caused by clonidine or adenosine. At a stimulation frequency of 5 Hz, the α 2-adrenoceptor antagonist idazoxan (0.1 μM) and the prostaglandin synthesis indomethacin (10 μM) both facilatated 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 α 1-adrenoceptor inhibition of noradrenaline release, but not in α 2-adrenoceptor, adenosine or prostaglandin inhibition.

Inhibition of noradrenaline release by neuropeptide Y in mouse atria does not involve inhibition of adenylate cyclase or a pertussis toxin-susceptible G protein

Neuropeptide Y (30-1000 nmol/l) significantly inhibited the fractional stimulation-induced outflow of radioactivity from mouse atria preincubated with [3H]-noradrenaline. The inhibitory effect of neuropeptide Y was observed at all frequencies tested (2, 5 and 10 Hz) as well as after alpha-adrenoceptor blockade with phentolamine (1 mumol/l). A combination of 8-bromo adenosine cyclic-3'-5'-monophosphate (90 or 270 mumol/l) with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100 mumol/l) was used to saturate maximally the adenylate cyclase system and these drug combinations significantly enhanced the stimulation-induced outflow of radioactivity. However, neuropeptide Y inhibited the stimulation-induced outflow in the presence of these drugs, suggesting that the inhibitory effect of neuropeptide Y was not due to decreasing endogenous cyclic AMP formation. Finally, atria from mice treated with pertussis toxin were used. In this case, the inhibitory effect of neuropeptide Y on the stimulation-induced outflow of radioactivity was still observed suggesting that inhibitory prejunctional neuropeptide Y receptors are not coupled to a pertussis toxin-susceptible G protein.

Facilitation of noradrenaline release by isoprenaline in rat isolated atria does not involve angiotensin II formation.

1. Rat isolated atria were incubated with 3H-noradrenaline and the intramural sympathetic nerves were stimulated at 2 Hz for 60 s. The stimulation-induced (SI) efflux of radioactivity was used as an index of release of transmitter noradrenaline. 2. Isoprenaline (0.1 mumol/L) alone did not increase noradrenaline release. Cocaine (30 mumol/L) produced a 73% increase in the stimulation-induced release of noradrenaline. In the presence of cocaine, isoprenaline enhanced noradrenaline release by 22%. 3. In the presence of cocaine, both angiotensin I (0.3 mumol/L) and angiotensin II (0.3 mumol/L) produced almost two-fold enhancements in the SI release of noradrenaline. 4. Captopril (5 mumol/L) blocked the facilitatory effect of angiotensin I on noradrenaline release but did not alter that of isoprenaline. 5. Saralasin (0.1 mumol/L) reduced the facilitatory effect of angiotensin II on noradrenaline release but did not alter that of isoprenaline. 6. The findings indicate that the facilitation of noradrenaline release by isoprenaline in rat atria is not mediated by local formation of angiotensin II.

Modulation of noradrenaline release in slices of rat kidney cortex through α1- and α2-adrenoceptors

Slices of rat kidney cortex were incubated in [ 3H]noradrenaline, then placed in a flow cell and subjected to electrical field stimulation. At a stimulation frequency of 5 Hz, both the α 2-adrenoceptor antagonist idazoxan (0.1 μM) and the α 1-adrenoceptor antagonist prazosin (0.1 μM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity from the slice. However, neither clonidine (0.1 μM) nor methoxamine (10 μM), α 2- and α 1-agonists respectively, affected the S-I outflow of radioactivity at this stimulation frequency. At a lower stimulation frequency (1 Hz), the S-I outflow of radioactivity was not affected by idazoxan or prazosin, but was inhibited by both clonidine and methoxamine. The effect of clonidine was prevented by idazoxan (0.1 μM), but not by prazosin (0.1 μM). The effect of methoxamine was abolished by prazosin (0.1 μM), but not by idazoxan (0.1 μM). The inhibitory effect of methoxamine was not prevented by the prostaglandin synthesis inhibitor indomethacin (10 μM) or the adenosine receptor antagonist 8-phenyltheophylline (1 μM) and thus was not mediated by either prostaglandins or adenosine. The results suggest that both prejunctional α 1- and α 2- adrenoceptors are directly involved in modulation of noradrenaline release from the renal sympathetic nerves of the rat.

Modulation of noradrenaline release by presynaptic alpha-2 and beta adrenoceptors in rat atria

The effect of pretreatment with the beta-2-selective adrenoceptor agonist, (+/-)-clenbuterol (0.3 mg/kg, twice daily, 14 days) on prejunctional alpha-2- and beta-adrenoceptors was studied in rat atria. When atria from non-pretreated rats had been preincubated with (3H)-noradrenaline, (-)-isoprenaline (0.02 to 4.0 microM) did not affect tritium overflow evoked by stimulation of the cardioaccelerant nerves, but a higher concentration (40 microM) decreased it. Blockade of prejunctional inhibitory alpha-2-adrenoceptors by yohimbine (0.03, 0.3 and 0.8 microM) enhanced the overflow of tritium. In the presence of yohimbine, isoprenaline (1.2 microM) significantly increased stimulation-induced transmitter overflow, suggesting that in rat atria the facilitatory effect of isoprenaline mediated via prejunctional beta-adrenoceptors, is masked by the dominant influence of inhibitory alpha-2-adrenoceptors. (-)-Propranolol (0.1 microM) prevented the isoprenaline-induced increase in atrial rate and the isoprenaline-induced enhancement of transmitter release in the presence of yohimbine (0.3 microM), but did not modify by itself the stimulation-induced efflux of tritium, suggesting that neuronally released noradrenaline failed to activate facilitatory prejunctional beta-adrenoceptors. When atria from clenbuterol-pretreated rats had been preincubated with 3H-noradrenaline, the facilitatory effect of yohimbine 0.03 and 0.3 microM was markedly enhanced and, in this case, isoprenaline (1.2 and 12.0 microM) failed to cause its facilitatory effect in the presence of the alpha-2-adrenoceptor antagonist. Propranolol did not modify the facilitatory effect of yohimbine. No changes in the isoprenaline-induced increase in atrial rate were observed in clenbuterol-treated rats.(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. It is probable that postjunctionally active PGF2 and PGI2 is formed locally from exogenous arachidonic acid, but not enough prejunctionally active PGE2 is synthesized to modulate renal transmitter release.

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). 6. These results suggest that angiotensin II receptor modulation of noradrenaline release is not mediated through either a pertussis toxin sensitive guanine nucleotide-binding protein or activation of protein kinase C.

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. 4. In rabbit pulmonary artery, two concentrations of 8-Br-cyclic AMP, (270 or 540 microM), enhanced the S-I outflow of radioactivity in the presence of phentolamine (1 microM) to a similar extent. In the presence of 8-Br-cyclic AMP (270 microM) and phentolamine, the facilitatory effects of isoprenaline (0.1 microM) and of ACTH,24 (0.1 microM) were blocked, whereas that of angiotensin II (0.01 microM) was not changed. These results suggest that both prejunctional beta-adrenoceptors and ACTH receptors enhance noradrenaline release by generating cyclic AMP. The mechanism by which angiotensin II facilitates noradrenaline release is probably independent of the cyclic AMP second messenger pathway.

The influence of activation or inhibition of protein kinase C on the release of radioactivity from rat isolated atria labelled with [3H]-noradrenaline.

1. The release of radioactivity from rat isolated atria preloaded with [3H]-noradrenaline ([3H]-NA) evoked by electrical field stimulation (2 Hz, 1 ms, 60 s) of intraneuronal sympathetic nerves, high potassium (64.7 mM) or tyramine (0.3 micron) was used as an index of noradrenaline release. 2. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) produced a concentration-dependent enhancement of field stimulation-induced outflow of radioactivity, whereas polymyxin B, an inhibitor of protein kinase C, reduced [3H]-NA release evoked by field stimulation. The enhancement observed in the presence of PMA was attenuated by polymyxin B (10 and 70 microns). 3. Release of noradrenaline evoked by membrane depolarization in a high potassium medium was similarly affected by PMA and polymyxin B. 4. In contrast, the release of noradrenaline evoked by the indirectly acting sympathomimetic amine, tyramine, was not altered by PMA. Polymyxin B in a concentration of 70 microns, but not 10 microns caused a slight reduction in tyramine-induced outflow of radioactivity. 5. The spontaneous outflow of radioactive compounds was not affected by either PMA or polymyxin B in the bathing medium. 6. The findings suggest that protein kinase C may play a role in the exocytotic release of noradrenaline but not in the displacement of noradrenaline by indirectly acting sympathomimetic amines.

Involvement of cyclic nucleotides in prejunctional modulation of noradrenaline release in mouse atria.

1 In mouse isolated atria previously incubated with [3H]-noradrenaline, 8-bromo-cyclic AMP (3-270 microM) produced a concentration-dependent increase in the fractional stimulation-induced outflow of radioactivity. 8-Bromo-cyclic GMP induced a lesser increase in the stimulation-induced outflow. 2 The phosphodiesterase inhibitors: M&B 22948 (90 microM); ICI 63197 (30 and 90 microM) and 3-isobutyl-1-methylxanthine (90 microM) increased the fractional stimulation-induced outflow. Together these results indicate that cyclic AMP may have a modulatory effect on noradrenaline release. 3 The inhibition of the stimulation-induced outflow produced by clonidine (0.03 microM) and its facilitation produced by phentolamine (1 microM) were unaltered in the presence of 8-bromo-cyclic AMP (90 microM). However, in the presence of 8-bromo-cyclic AMP (270 microM), the facilitatory effect of phentolamine was enhanced, but the inhibitory effect of clonidine (0.03 microM) was unaltered. In the presence of ICI 63197 (30 microM) the inhibitory effect of clonidine (0.03 microM) was unaltered, but the facilitatory effect of phentolamine (1 microM) was slightly enhanced. 4 Isoprenaline (0.003-0.1 microM) enhanced the fractional stimulation-induced outflow, an effect blocked by propranolol (0.1 microM). In the presence of 8-bromo-cyclic AMP (90 microM), the facilitatory effect of isoprenaline (0.01 microM) was blocked. In the presence of ICI 63197 (30 microM) the facilitatory effect of isoprenaline (0.003 microM) was potentiated. 5 These results suggest that whereas beta-adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase and enhanced formation of cyclic AMP, alpha-adrenoceptor-mediated inhibition of noradrenaline release is not linked to inhibition of adenylate cyclase activity.

Modulation of Norepinephrine Release Through α1 and α2-Adrenoceptors in Rat Isolated Kidney

The aim of this study was to investigate alpha-adrenoceptor modulation of norepinephrine (NE) release from sympathetic nerves in rat isolated perfused kidney. After preincubation with [3H]NE, the renal nerves were stimulated. The stimulation-induced (S-I) outflow of radioactivity was used as an index of NE release. Clonidine (0.1 mumol/L) decreased the S-I outflow of radioactivity. This effect was abolished by the alpha 1-adrenoceptor antagonist idazoxan (0.1 mumol/L) but not by the alpha 2-adrenoceptor antagonist prazosin (0.1 mumol/L). Methoxamine (10 mumol/L) also had an inhibitory effect; this was abolished by prazosin (0.1 mumol/L), but not by idazoxan. Individually, these alpha-blocking drugs and the alpha 1-adrenoceptor antagonist corynanthine (0.3 mumol/L) enhanced S-I outflow of radioactivity. In the presence of indomethacin (10 mumol/L), the inhibitory effect of methoxamine was abolished but clonidine still inhibited S-I outflow of radioactivity. The facilitatory effect of prazosin was also unaltered by indomethacin. These results suggest the existence of inhibitory prejunctional alpha 1- and alpha 2-adrenoceptors in the kidney. The inhibitory effect of methoxamine seems to be mediated through prostaglandin inhibition of NE release. However, the evidence for inhibitory prejunctional alpha 1-adrenoceptors rests solely on the facilitatory effects of prazosin and corynanthine.