Modulation of noradrenaline release in slices of rat kidney cortex through alpha 1- and alpha 2-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 alpha 2-adrenoceptor antagonist idazoxan (0.1 microM) and the alpha 1-adrenoceptor antagonist prazosin (0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity from the slice. However, neither clonidine (0.1 microM) nor methoxamine (10 microM), alpha 2- and alpha 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 microM), but not by prazosin (0.1 microM). The effect of methoxamine was abolished by prazosin (0.1 microM), but not by idazoxan (0.1 microM). The inhibitory effect of methoxamine was not prevented by the prostaglandin synthesis inhibitor indomethacin (10 microM) or the adenosine receptor antagonist 8-phenyltheophylline (1 microM) and thus was not mediated by either prostaglandins or adenosine. The results suggest that both prejunctional alpha 1- and alpha 2-adrenoceptors are directly involved in modulation of noradrenaline release from the renal sympathetic nerves of the rat.     

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.     

Modulation of norepinephrine release through alpha 1- and alpha 2-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.     

Alpha 2-adrenoceptor activation inhibits noradrenaline release in human and rabbit isolated renal arteries.

The aim of the present study was to investigate alpha 2-adrenoceptor modulation of noradrenaline release in superfused 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 alpha 2-adrenoceptor agonist clonidine (0.1 mumol/l) failed to inhibit stimulation-induced outflow of radioactivity in human and rabbit renal arteries whereas the alpha 2-adrenoceptor agonist UK 14304 (0.1 mumol/l) did inhibit stimulation-induced outflow. The inhibitory effect of UK 14304 in human renal arteries was blocked by the alpha 2-adrenoceptor blocking drug rauwolscine (1 mumol/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 alpha 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 alpha 2-adrenoceptors. At a higher stimulation frequency most of the prejunctional alpha 2-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 alpha 2-adrenoceptors.     

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)     

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)     

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)     

Presynaptic alpha 2-adrenoceptor antagonism by verapamil but not by diltiazem in rabbit hypothalamic slices.

1 Rabbit hypothalamic slices prelabelled with [3H]-noradrenaline and superfused with Krebs solution were stimulated electrically at a frequency of 5 Hz. Exposure to verapamil (0.1 to 10 microM) significantly increased, in a concentration-dependent manner, the electrically-evoked overflow of tritium, without affecting the spontaneous outflow of radioactivity. 2 Exposure to diltiazem in concentrations up to 100 microM had no effect on the electrically evoked release of [3H]-noradrenaline, but increased the basal outflow of radioactivity at 10 and 100 microM. 3 The preferential alpha 2-adrenoceptor antagonist, yohimbine (0.1 microM) significantly antagonized the inhibitory effect of clonidine or adrenaline on [3H]-noradrenaline overflow elicited by electrical stimulation. Verapamil (3 microM) also antagonized this inhibitory effect of the alpha 2-adrenoceptor agonists on [3H]-noradrenaline release. In contrast to these results, exposure to diltiazem (10 microM) was ineffective in blocking the action of the alpha 2-adrenoceptor agonist. 4 These results suggest that the two Ca2+-antagonists verapamil and diltiazem differ in their ability to affect central noradrenergic neurotransmission. While verapamil is a relatively potent alpha 2-adrenoceptor antagonist, diltiazem is devoid of presynaptic alpha 2-adrenoceptor antagonist properties.     

Prejunctional alpha 1-adrenoceptors modify release of [3H]noradrenaline in the guinea-pig vas deferens

1. Several alpha 1- and alpha 2-adrenoceptor agonists and antagonists were examined for effects on spontaneous and stimulus-evoked release of [3H]noradrenaline from sympathetic nerves in guinea-pig vas deferens. 2. Prazosin (0.1 and 1 microM), phentolamine (30 microM) and yohimbine (10 microM) each enhanced the stimulus-evoked release of [3H]noradrenaline. 3. Prazosin and phentolamine increased the spontaneous outflow of [3H]noradrenaline, whereas yohimbine was without effect. 4. Methoxamine (10 microM) and clonidine (0.1 microM) inhibited the stimulus-evoked release of [3H]noradrenaline, whereas only methoxamine (1 microM) decreased the spontaneous outflow of [3H]noradrenaline. 5. The identity of prejunctional alpha-adrenoceptors in the guinea-pig vas deferens is discussed.     

Pharmacological characterization of presynaptic alpha-adrenoceptors in the modulation of the 5-hydroxytryptamine release from vascular adrenergic nerves in the rat

The role of presynaptic alpha-adrenoceptors in modulation of the 5-hydroxytryptamine (5-HT) release from vascular adrenergic nerves was investigated in the perfused mesenteric vascular bed of the rat. After treatment with 5-HT (10 microM) for 15 min, the vasoconstrictor response to periarterial nerve stimulation (PNS, 4 to 16 Hz, 2 msec in duration for 30 sec) was greatly potentiated without significantly affecting the pressor response to exogenously administered noradrenaline (0.5 nmol). The potentiating effect was more pronounced at low frequencies of PNS (4 and 8 Hz). The potentiation of the pressor response to PNS after 5-HT treatment did not occur in the presence of LY53857 (0.01 microM), a selective 5-HT2 receptor antagonist. The enhanced pressor response to PNS seen after 5-HT treatment was further exaggerated in the presence of clonidine (0.1 and 1 microM), a preferential alpha 2-adrenoceptor agonist, while methoxamine (1 and 10 microM), a selective alpha 1-adrenoceptor agonist, did not affect the enhanced PNS response. This effect of clonidine was more pronounced in low frequencies of PNS (4 and 8 Hz) and was abolished by LY53857 (0.01 microM). In the perfused mesenteric vascular bed labelled with [3H]-5-HT, PNS (8 Hz) evoked an increase of tritium efflux in the perfusate. The PNS-evoked tritium efflux was facilitated by yohimbine (0.1 to 1 microM), an alpha 2-adrenoceptor antagonist, and prazosin, a selective alpha 1-adrenoceptor antagonist, at a high concentration (1 microM), while LY53857 (0.01 to 0.1 microM) and a low concentration of prazosin (0.1 microM) had no effect on the tritium efflux. Clonidine (0.01 to 1 microM) produced a dose-dependent increase of PNS-evoked tritium efflux, while methoxamine (0.1 to 10 microM) was without effect. The monoamine uptake inhibitor, cocaine (10 microM) produced a significant inhibition of the PNS-evoked tritium efflux. The effects of clonidine and cocaine on the PNS-evoked tritium efflux were antagonized by yohimbine (1 microM). These results suggest that the release of 5-HT from adrenergic nerve endings by PNS is modulated by presynaptic alpha 2-adrenoceptors.     

Noradrenaline stimulates 5-hydroxytryptamine release from mouse ileal tissues via alpha(2)-adrenoceptors.

The effect of noradrenaline on 5-hydroxytryptamine (5-HT) release from isolated mouse ileal tissues was investigated. Noradrenaline, but not isoprenaline, at 1 microM stimulated 5-HT release, an effect which was inhibited by yohimbine, an alpha(2)-adrenoceptor antagonist, but not by bunazosin, an alpha(1)-adrenoceptor antagonist. alpha(2)-Adrenoceptor agonists, UK 14,304 (5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline) and clonidine at a higher concentration (10 microM) also stimulated 5-HT release, while alpha(1)-adrenoceptor agonists, methoxamine and phenylephrine, had no effect. The effect of noradrenaline was completely abolished in ileal tissues isolated from mouse treated with pertussis toxin (100 microg/kg, i.v.) for 2 days. These results suggest that noradrenaline causes 5-HT release from enterochromaffin cells in mouse ileal tissues via alpha(2)-adrenoceptor subtypes coupled to a pertussis toxin-sensitive G protein.     

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)     

Interactions between the effects of endothelin-1, clonidine and yohimbine on electrically-induced contractions in rat vas deferens.

1. The relationship between endothelin-1(ET-1)-induced effects on the contractile responses of epididymal portion of rat vas deferens elicited by field electrical stimulation (FES: 80 V, 1 msec, 0.1 Hz) and the effects of the alpha 2-adrenoceptor agonist clonidine and the alpha 2-adrenoceptor antagonist yohimbine were studied. 2. ET-1 (0.01 nM-0.1 microM) concentration-dependently increased the FES-induced contractions. 3. ET-1 (0.1 nM-0.1 microM) reversed the inhibitory effect of clonidine on the FES-evoked contractions whereas ET-1 applied before clonidine exerted a dual effect on the clonidine-induced inhibition of the FES-evoked contractions. 4. The ET-1-induced enhancement of FES-induced contractions was potentiated in the presence of 1 microM yohimbine and was not observed at all in the presence of 10 microM yohimbine. Yohimbine, applied at concentrations of 1 and 10 microM exerted similar blocking effects on the alpha 1-adrenoceptor agonistic effects of phenylephrine. However, yohimbine at a concentration of 10 microM markedly potentiated the contractile effect of exogenous adenosine 5'-triphosphate (ATP), 30 microM. Tetrodotoxin abolished this effect of yohimbine. 5. The results presented here suggest the existence of modulating interactions between the ET-1-evoked increase of FES-induced contractions of rat vas deferens and the alpha 2-adrenoceptor drugs clonidine and yohimbine.     

The effect of alpha 2-adrenoceptor agonists on the acid secretory responses of rat isolated gastric mucosa to electrical field stimulation.

The effects of clonidine, UK-14,304, noradrenaline, para-aminoclonidine and phenylephrine were examined on the acid secretory response of the rat isolated gastric mucosa preparation to electrical field stimulation. Clonidine, UK-14,304, noradrenaline and para-aminoclonidine but not phenylephrine (10 microM) reduced the response of the gastric mucosa stimulated at 2.5 Hz; gastric mucosae stimulated at higher frequencies were insensitive to the action of these alpha 2-adrenoceptor agonists. The inhibitory effect of the selective alpha 2-adrenoceptor agonist UK-14,304 was antagonized by idazoxan but not by prazosin. These findings indicate that clonidine and other alpha 2-adrenoceptor agents inhibit the acid secretory response of the rat gastric mucosa to electrical field stimulation by an action at alpha 2-adrenoceptors, which are probably located on cholinergic nerve terminals.     

Adenosine A(1) receptor stimulation inhibits alpha(1)-adrenergic activation of the cardiac sarcolemmal Na(+)/H(+) exchanger

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is increased by stimulation of G(q) protein-coupled receptors (G(q)PCRs), but the roles of other GPCRs are largely unknown. We determined the effects of N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236), a selective agonist of the G(i)PCR adenosine A(1) receptor, on sarcolemmal NHE activity in adult rat ventricular myocytes (n=8-10 per group). NHE activity was indexed by the H(+) efflux rate after intracellular acidification, measured by microepifluorescence. GR79236 alone (0.01-10 microM) had no effect on NHE activity. However, co-administration of GR79236 inhibited, in a concentration-dependent manner, the stimulation of NHE activity by the alpha(1)-adrenoceptor agonist phenylephrine (10 microM). The inhibitory effect of GR79236 (10 microM) was abolished by (1) the selective A(1) antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.1 microM), confirming an A(1) receptor-mediated action, and (2) pre-treatment with pertussis toxin (5 microgram ml(-1) for 60 min), indicating a G(i) protein-mediated mechanism. Our data suggest the existence of inhibitory crosstalk between the G(i)PCR adenosine A(1) receptor and the G(q)PCR alpha(1)-adrenoceptor in the regulation of sarcolemmal NHE activity.     

Changes in neuromuscular transmission of guinea pig vas deferens produced by decamethrin treatment

Responses of the isolated vas deferens of guinea pig to clonidine (inhibition of contractions to field stimulation at 2.5 Hz), tyramine (inhibition of contractions to field stimulation at 10 Hz), prostaglandin E2 (inhibition of contractions to field stimulation at 10 Hz), and noradrenaline (contraction of longitudinal muscle) were determined after administration of decamethrin (18 mg kg-1, ip) once a day for 3 consecutive days. Treatment with decamethrin produced a subsensitivity of the prejunctional alpha 2-adrenoceptor system as evidenced by the fact that the alpha 2-agonist clonidine was less effective in decreasing nerve-stimulated induced contractions of the vas deferens. In addition, the presynaptic action of tyramine on postganglionic motor transmission was impaired. However, no detectable changes in the inhibition by prostaglandin E2 of twitch responses were produced by decamethrin. Decamethrin treatment had a significant effect on noradrenaline responsiveness, causing an increase in the maximum contractile response, indicative of an enhanced postreceptor mechanism. The present results suggest that decamethrin treatment reduces peripheral presynaptic adrenoceptor sensitivity. This reduction will lead subsequently to increased noradrenaline release and postsynaptic adrenoceptor upregulation.     

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)     

Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes

1. Human alpha(2A)-adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand-induced extracellular acidification rate changes. 2. In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 microM. In alpha(2A)-adrenoceptor transfected cells the rank order of agonist potency was A-54741 (mean pEC(50)=8.96)>dexmedetomidine (8.88)>UK-14304 (8.42)>B-HT 920 (7.05)>noradrenaline (6.92). A-54741, UK-14304 and noradrenaline had the same maximum response while dexmedetomidine and B-HT 920 behaved as partial agonists. 3. The selective alpha(2)-adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pK(B)) against noradrenaline was 8.43. 4. The selective alpha(1)-adrenoceptor ligands prazosin and doxazosin (each 3 microM) had no effect on noradrenaline responses. 5. Acidification rate changes induced by each agonist were abolished by pre-treatment of cells with pertussis toxin. 6. These data suggest that agonist-induced acidification rate responses in CHL cells transfected with the human alpha(2A)-adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive G(i/o) proteins.     

Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.

1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3. Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3'',5''-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8-chloroadenosine 3'',5''-cyclic monophosphorothioate (RClcAMPS, 300 microM), suggesting that PKA activation enhances basal noradrenaline biosynthesis in sympathetic nerve terminals. 5. The protein kinase inhibitors, KN-62 (CAM kinase II, 10 microM), RClcAMPS (PKA, 300 microM), polymyxin B (protein kinase C (PKC), 21 microM) and staurosporine (PKC, PKA and CAM kinase II, (0.1 microM) did not affect S-I synthesis, although KN-62, polymyxin B and staurosporine decreased S-I release. We conclude that S-I synthesis is triggered by Ca2+ entering the neurone but that the signalling pathway does not involve classical protein kinases and appears distinct from the steps involved in transmitter release.     

Differential blocking actions of idazoxan against the inhibitory effects of 6-fluoronoradrenaline and clonidine in the rat vas deferens

The prejunctional inhibitory effects of clonidine and 6-fluoronoradrenaline (6-FNA) have been evaluated in the isolated prostatic segment of the rat vas deferens, against the twitch response evoked by low frequency (0.1 Hz) field stimulation. The inhibitory potency of 6FNA was significantly increased in the presence of cocaine (1 microM) or pargyline (10 microM), but was not modified in the vas deferens from rats pretreated with reserpine when the endogenous levels of noradrenaline (NA) were decreased by 97%. Clonidine was significantly more potent than 6-FNA as an inhibitory agonist, and the potency of clonidine was not modified after cocaine, pargyline or reserpine. The alpha 2-adrenoceptor blocking agent idazoxan, was a competitive antagonist against the inhibitory effects of clonidine under all experimental conditions. In contrast, the only antagonism shown by idazoxan against the inhibitory effects of 6-FNA was in the presence of cocaine (1 microM), and this antagonist effect of idazoxan was not concentration-related. Low concentrations of 6-FNA caused concentration-dependent facilitatory effects on the twitch response, which were significantly greater after treatment with idazoxan (1 microM) in reserpine-treated vas deferens. These facilitatory effects of 6-FNA were always observed in the presence of prazosin (300 nM) and also after treatment of the preparations with phenoxybenzamine (10 microM), a concentration which abolished the inhibitory actions of both clonidine and 6-FNA. The facilitatory effects on the twitch response induced by low concentrations of 6-FNA are therefore unlikely to be due to either alpha 1- or alpha 2-adrenoceptor stimulation. In conclusion, the failure of idazoxan to block the inhibitory effects of 6-FNA, while exerting a potent competitive antagonism of clonidine-induced inhibitory effects, supports the proposal that alpha 2-adrenoceptors may in fact be subdivided into two subclasses, involving imidazoline and phenylethylamine recognition sites.