Modulatory effect of bradykinin on noradrenaline release in isolated atria from normal and B2 knockout transgenic mice.

The modulatory effect of bradykinin on electrically-induced noradrenaline release was assessed in isolated atria from normal and B2 knockout transgenic mice preincubated with [3H]noradrenaline. Concentrations of 1, 3 and 10 nM of bradykinin did not significantly alter the outflow of radioactivity whereas higher concentrations of bradykinin (30 and 100 nM) enhanced it. The facilitatory effect of 30 nM bradykinin was inhibited by a selective bradykinin B2 receptor antagonist. Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, 30 nM), and by a protein kinase C inhibitor, bisindolylmaleimide (1 microM). The co-administration of bradykinin (1 to 100 nM) with either [Leu8]des-Arg9-bradykinin (100 nM), AcLys[DbetaNal7,Ile8]des-Arg9-bradykinin (30 nM) (bradykinin B1 receptor antagonists) or diclofenac (1 microM) (a cyclooxygenase inhibitor), shifted the facilitatory effect of bradykinin to lower concentrations. The facilitatory effect of bradykinin also was enhanced by enalaprilat (1 microM) and mergetpa (1 microM), inhibitors of angiotensin-converting enzyme (kininase II) and kininase I, respectively. In contrast, selective bradykinin B1 receptor agonists, des-Arg9-bradykinin (1 to 100 nM) and Sar[D-Phe8]des-Arg7-bradykinin (1 to 100 nM), did not significantly affect the stimulation-induced outflow of radioactivity. Neither bradykinin (100 nM) nor des-Arg9-bradykinin (100 nM) had any modulatory effect in B2 knockout transgenic mice. These findings suggest that the facilitatory effect of bradykinin on noradrenaline release in the mouse atria is mediated exclusively by presynaptic bradykinin B2 receptors which are linked to protein kinase C. The greater release of noradrenaline with bradykinin under inhibition of prostaglandins production and kininases I and II activity might be of importance in pharmacotherapies.

Impaired basal sympathetic tone and alpha1-adrenergic responsiveness in association with the hypotensive effect of melatonin in spontaneously hypertensive rats.

Early investigations have suggested a relationship between hypertension and melatonin, a pineal hormone. The aims of this study were to evaluate the implication of the sympathetic nervous system in the acute effect of melatonin on blood pressure in conscious 12-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), and to determine whether the hypotensive effect of melatonin is associated with alterations in pre- or postsynaptic mechanisms. Melatonin, 10 mg/kg, produced a sustained time-dependent decrease of mean arterial pressure only in SHR without changes in heart rate in both groups. Until 20 min after melatonin administration, plasma epinephrine (EPI) levels were reduced by about 60% in both groups, but norepinephrine (NE) levels were decreased only in SHR by about 30%. The nitroprusside-induced hypotension responses and the associated increases in heart rate were similar in both groups before or after administration of melatonin. Unexpectedly, the sympathetic reactivity to nitroprusside, evaluated by the increases in NE and EPI, was markedly enhanced after melatonin treatment in both WKY and SHR. The stimulation induced [3H]-norepinephrine release from isolated atria was not altered by melatonin in SHR. In cultured aortic vascular smooth muscle cells, the basal and phenylephrine induced inositol phosphate formations were greater in SHR, and the melatonin pretreatment dose dependently attenuated the phenylephrine responses in cells from both WKY and SHR. Therefore the hypotensive action of melatonin appears to be associated with an inhibition of basal sympathoadrenal tone and could also be mediated partly by the blockade of postsynaptic alpha1-adrenergic receptor-induced inositol phosphate formation.

Sympathetic functions in NG-nitro-L-arginine-methyl-ester-induced hypertension: modulation by the renin-angiotensin system.

BACKGROUND: Nitric oxide and angiotensin II have been shown to attenuate cardiac beta-adrenergic inotropism. OBJECTIVE: To study sympathetic presynaptic and post-synaptic functions after chronic nitric oxide synthesis blockade with NG-nitro-L-arginine-methyl-ester (L-NAME, for 40 days) in association with renin-angiotensin system blockade (during the last 12 days) in order to evaluate the possible physiological interactions between these systems. METHODS: Haemodynamic parameters in conscious rats were assessed. Release of noradrenaline from isolated atria and cardiac beta-adrenergic-adenylyl cyclase pathway in rats of sham-treated and L-NAME-treated groups, with or without losartan or enalaprilat treatment, were assessed. RESULTS: L-NAME-treated rats developed a time-dependent increase in blood pressure associated with increased plasma adrenaline levels whereas plasma noradrenaline and cardiac catecholamine levels were similar to those in sham-treated rats. Field-stimulated release of noradrenaline, cardiac beta-adrenoceptor density and affinity and isoproterenol-stimulated formation of cyclic AMP were similar in sham and L-NAME-treated rats. However, Gpp(NH)p, NaF and forskolin-stimulated adenylyl cyclase activity were greater in L-NAME rats although Gs and Gi protein levels were similar in sham-treated and L-NAME-treated rats. Losartan and enalaprilat treatments exerted equipotent angiotensin-pressor response blockade and hypotensive effects whereas catecholamine levels were not altered. Interestingly, only losartan treatment acted to reduce the increased Gs-adenylyl cyclase activity in L-NAME rats, without alteration of G protein levels. CONCLUSIONS: The nitric oxide synthase blockade-induced hypertension seems to be associated with increased adrenal-medullary system and renin-angiotensin system activities. The increased Gs-adenylyl cyclase activity after chronic inhibition of formation of nitric oxide suggests that nitric oxide plays a modulatory role in formation of cyclic AMP, to which angiotensin II seems to contribute through an angiotensin II type 1 receptor-mediated mechanism.

Modulation of noradrenaline release by B1 and B2 kinin receptors during metabolic anoxia in the rat isolated atria.

A model of metabolic anoxia was used to investigate the modulatory effect of bradykinin (BK) on the release of noradrenaline (NA) in isolated rat atria. Atria were isolated from Wistar rats and inserted into a perfusion system. After an equilibration period of 20 min, the perfusate was collected every 5 min for a period of 85 min, during which the atria were field stimulated (5 Hz, 2 ms, 50 mA, 60 s) at 10 (S1) and 75 (S2) min. The metabolic anoxia was started 40 min before S2 by replacing O2 with N2 and by removing glucose. The drugs were added 20 min before S2, and their effects on NA release were assessed by the ratio S2/S1. The spontaneous and electrically stimulated induced (S-I) releases of NA were significantly increased by the anoxic procedure. BK (30 nM) significantly increased the S-I release of NA under normoxic conditions. However, under anoxia, BK had no effect on the S-I release of NA but inhibited its spontaneous release. BK coadministered with HOE-140 (100 nM), a B2 receptor antagonist, significantly increased the S-I release of NA during anoxia, whereas the coadministration of BK with Leu3-des-Arg9-BK (100 nM), a B1 receptor antagonist, significantly inhibited that release. Administration of des-Arg9-BK (100 nM) had no effect on the S-I outflow of NA following anoxia, although its coadministration with a B1 antagonist resulted in a significant inhibition of the S-I outflow of NA. The present results suggest that BK inhibits NA release through the activation of a B2 receptor following a 40-min period of metabolic anoxia. Because this inhibition can be observed only in the presence of a B1 receptor antagonist, this could imply that B1 receptor activation, revealed by the anoxia, is involved in the facilitation of NA release.

Paradoxical action of desipramine on the modulatory effect of bradykinin on noradrenaline release in a model of metabolic anoxia in rat isolated atria.

We have previously shown that bradykinin (BK) can modulate the release of noradrenaline (NA) in a model of metabolic anoxia in the rat isolated atria. In this study, we tested the effect of an inhibitor of NA reuptake, desipramine, on the modulatory action of BK on NA release in this experimental model. Atria were isolated from Wistar rats and inserted into a perfusion system. After an equilibration period of 20 min, the perfusate was collected every 5 min for a period of 85 min, during which the atria were field stimulated (5 Hz, 2 ms, 50 mA, 60 s) at 10 (S1) and 75 (S2) min. Desipramine (1 microM) was present throughout the experimental procedures. The metabolic anoxia was started 40 min before S2 by replacing O2 with N2 and by removing glucose. The drugs were added 20 min before S2, and their effects were assessed by the ratio S2/S1. The spontaneous release of NA was not changed by the anoxic procedure, which significantly increased the electrically stimulated induced (S-I) release of NA. BK (30 nM) significantly increased the S-I release of norepinephrine under normoxic conditions. However, following anoxia, both BK and the B1 receptor agonist des-Arg9-BK (100 nM) significantly inhibited the S-I release of NA. The inhibition induced by BK was prevented by selective antagonists for B1 and B2 receptors. These observations contrast with the results obtained without desipramine, where BK, but not des-Arg(9)-BK, inhibited the S-I release of NA during anoxia. Therefore, blockade of NA reuptake during metabolic anoxia appears to alter the modulatory effect of kinins on NA release via the B1 receptor in the rat isolated atria.

Transient ischemia inhibits nonexocytotic release of norepinephrine following sustained ischemia in rat heart: is bradykinin involved?

Previous studies have demonstrated that transient ischemia inhibits the release of norepinephrine (NE) following a sustained ischemia. However, the mechanism underlying this inhibition is unknown. Therefore, this study was designed to investigate whether bradykinin (BK) may be involved in the inhibition of NE release following ischemic preconditioning. The effects of transient ischemia, exogenous BK, and kinin receptor blockers on NE release after a prolonged ischemia were tested in the isolated rat heart preparation. Three cycles of 5-min ischemia and reperfusion resulted in the reduction of NE release from 115.3 +/- 14.5 to 51.6 +/- 9.3 pmol.g-1 (p < 0.05) after 30 min of subtotal global ischemia. This effect was not prevented by the administration of either Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), a B1 antagonist, or HOE-140 (1 mumol.L-1), a B2 antagonist. Three cycles of 5-min BK or des-Arg9-BK infusion also resulted in a dose-dependent inhibition of NE release after 30 min of ischemia. The inhibitory effects of BK (1 mumol.L-1) or des-Arg9-BK (0.5 mumol.L-1) were blocked by Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), but not by HOE-140 (1 mumol.L-1). The results show that transient ischemia and BK protect sympathetic nerve endings in the isolated rat heart. The inhibition of NE release by pretreatment with BK is mediated by the activation of B1 receptors, whereas preconditioning provided by transient ischemia may be mediated by a different, yet unknown, mechanism in the rat heart.

Interleukin-1 beta and tumor necrosis factor-alpha inhibit the release of [3H]-noradrenaline from isolated human atrial appendages.

In the present study, we have investigated the ability of human recombinant interleukin-1 beta (hIL-1 beta) and human recombinant tumor necrosis factor-alpha (hTNF-alpha) to modulate the stimulation-induced (S-I) outflow of [3H]-noradrenaline ([3H]-NA) from isolated superfused human atria. Pieces of human right atrial appendages were excised during routine cardiac surgery. Tissues were incubated with [3H]-NA (0.2 mumol/l) for 30 min at 37 degrees C, then inserted in a Brandel suprafusion system where the radioactivity was washed for 75 min with a Krebs-Henseleit solution at a rate of 0.4 ml/min. Thereafter, the effluent was collected for the remainder of the protocol during which two trains of electrical stimulation (50 mA intensity, 5 Hz frequency, 60 s duration, 2 ms pulses) were delivered at 10 min and 45 min (short protocol) or 85 min (long protocol). The effect of drugs on the S-I outflow of [3H]-NA was determined by adding drugs 20 min (short protocol) or 60 min (long protocol) before the second stimulation. Experiments were carried out in the continuous presence of desipramine (1 mumol/l) to prevent neuronal NA reuptake. The results showed that in human atrium, hIL-1 beta (3 ng/ml) and hTNF-alpha (0.5 ng/ml) significantly inhibited the S-I release of [3H]-NA. The inhibitory effect of hIL-1 beta was blocked by human recombinant IL-1 receptor antagonist (50 ng/ml), and by the cyclooxygenase inhibitor, diclofenac (1 mumol/l), suggesting that hIL-1 beta inhibited NA release through the formation of prostaglandins. The ability of hIL-1 beta and hTNF-alpha to inhibit NA release suggest that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system in human atrial appendages.

Modulation of noradrenaline release from isolated human atrial appendages.

Prejunctional modulation of noradrenaline release has been studied extensively in various experimental preparations. However, the presence and importance of prejunctional noradrenaline release modulation in human cardiac tissue is still unclear. In this study, we have used superfused human right atrial appendages excised from patients undergoing open heart surgery. The tissues were cut into six pieces and incubated with [3H]noradrenaline (4 microCi/ml. 0.2 microM) for 30 min at 37 degrees C. The tissues were then inserted into a suprafusion system and washed for 75 min with a Krebs-Henseleit solution at a rate of 0.4 ml/min. The experimental protocol consisted of a 60-min perfusion period during which a field stimulation (2 ms pulses, 60 s. 50 mA, 5 Hz) was delivered at 10 and 45 min. The effect of the drugs on the stimulation-induced outflow of radioactivity was determined by adding them 20 min before the second stimulation. Each experiment was carried out with or without desipramine (1 microM) to study the influence of the reuptake blockade. Fenoterol (1-1000 nM), a beta 2-adrenoceptor agonist, and angiotensin II (1-1000 nM) significantly increased noradrenaline release in a concentration-dependent manner. The administration of arecaidine propargyl ester (0.03-3 microM), a non-specific muscarinic receptor agonist, and propylnorapomorphine (0.1 nM-1 microM), a DA2-dopaminergic agonist, produced a concentration-dependent inhibition of the stimulation-induced outflow of radioactivity. The alpha 2-adrenoceptor agonist, oxymetazoline (1 microM) inhibited noradrenaline release at a stimulation frequency of 2 Hz, but not at 5 and 10 Hz. The alpha 2-adrenoceptor antagonist, idazoxan (1 microM), significantly increased the release of noradrenaline at 2 and 5 Hz but not at 10 Hz. The results obtained in the present study demonstrated the presence of the facilitatory beta 2-adrenoceptor and angiotensin II receptor as well as the presence of inhibitory alpha 2-adrenoceptor, muscarinic and DA2-dopamine receptors in the human atrial appendage.

Advancing age alters intracellular calcium buffering in rat adrenergic nerves.

There is a marked increase with advancing age of stimulation-evoked neurotransmitter release from vascular adrenergic nerves in the rat, an effect correlated with increased levels of plasma norepinephrine. This increase in norepinephrine release could not be accounted for by an alteration in neuronal and extraneuronal uptake of norepinephrine or a decline in feedback inhibition of release by prejunctional alpha2-adrenergic receptors. Measurement of intracellular calcium in fura-2-labeled superior cervical ganglion cells revealed elevated K+-evoked calcium transients in old compared to young neurons. Blockade of mitochondrial calcium uptake with dinitrophenol resulted in increased calcium transients in old neurons only. Furthermore, following blockade of mitochondrial calcium uptake the rate of return of calcium to resting levels was reduced to a greater degree in old cells as compared to young cells. The effects of dinitrophenol in old cells were attenuated when extracellular calcium was reduced. These findings suggest that older cells are more dependent on mitochondrial calcium buffering, perhaps due to changes in ATP dependent calcium uptake. Increased calcium transients as a result of altered intracellular calcium buffering offer a reasonable explanation for our previous observation of increased stimulation evoked norepinephrine release.

Interleukin-1 beta and tumor necrosis factor-alpha inhibit the release of [3H]-noradrenaline from mice isolated atria.

In the present study, we have investigated the ability of four cytokines, interleukin-1 beta, interleukin-2, interleukin-6 and tumor necrosis factor-alpha, to modulate the stimulation-induced outflow of radioactivity from isolated superfused mouse atria which where pre-incubated with [3H]-noradrenaline. The tissues were subjected twice to field stimulation (5 Hz frequency, 50 mA intensity, 2 ms pulses for 60 s) and the drugs were added prior to the second stimulation in order to assess their modulatory effects. The results show that mouse recombinant interleukin-1 beta and tumor necrosis factor-alpha inhibited the stimulation-induced release of radioactivity from the isolated mouse atria. The effect of interleukin-1 beta was blocked by a human recombinant interleukin-1 receptor antagonist. The inhibitory effect of interleukin-1 beta was also abolished by the cyclooxygenase inhibitor, diclofenac (1 mumol/l) suggesting that the action of interleukin-1 beta might be mediated through the formation of prostaglandins. The effect of interleukin-1 beta appears to be time-dependent, since a stronger inhibition of radio-activity release was observed when the incubation time was increased from 20 to 65 minutes before the second stimulation. Interleukin-2 and interleukin-6 were ineffective in modulating release under these experimental conditions. The ability of interleukin-1 beta and tumor necrosis factor-alpha to inhibit noradrenaline release suggests that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system.

Effects of chronic treatment with losartan and enalaprilat on [3H]-norepinephrine release from isolated atria of Wistar-Kyoto and spontaneously hypertensive rats.

The present study was designed to evaluate the effect of chronic treatment with losartan, an AT1 angiotensin II receptor antagonist, and enalaprilat, an angiotensin converting enzyme inhibitor, on the presynaptic modulation of [3H]-norepinephrine release from isolated atria of spontaneously hypertensive rats (SHR) and their respective control, the Wistar-Kyoto rats (WKY). The rats received either losartan (5 mg/kg/day) or enalaprilat (1 mg/kg/day) for 12 days by means of osmotic minipumps. The atria were isolated and incubated with [3H]-norepinephrine and the release of radioactivity was used as an index of norepinephrine release. The experimental protocol consisted of two electrical stimulations and the drugs were administered 20 min before the second stimulation. The modulatory action of angiotensin II (0.01 and 1 mumol/L), the alpha 2-adrenoceptor agonist, oxymetazoline (1 mumol/L), the alpha 2-adrenoceptor antagonist, idazoxan (1 mumol/L) and the beta 2-adrenoceptor agonist fenoterol (1 mumol/L) were tested. The results show that losartan or enalaprilat both similarly reduced the blood pressure in SHR. However, only the chronic losartan treatment, and not enalaprilat, abolished the facilitatory effect of exogenously administered angiotensin II on the release of radioactivity. The prejunctional alpha 2- and beta 2-adrenoceptor modulatory mechanisms were not altered by either chronic treatments. Similarly, the facilitatory effect of angiotensin II was blocked by acute administration of losartan but not by enalaprilat. Finally, the facilitatory action of bradykinin on the release of radioactivity was unchanged by chronic enalaprilat treatment. These results confirm the presence of facilitatory AT1 angiotensin II receptors on sympathetic nerve terminals of rat atria. These results also confirm that sympathetic nerve terminal blockade by losartan or the blockade of endogenous angiotensin II formation by enalaprilat are likely to participate in the antihypertensive action of AT1 angiotensin II receptor antagonists and angiotensin converting enzyme.

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.

Increase in [Ca2+]i by CCh in adult rat sympathetic neurons are not dependent on intracellular Ca2+ pools.

We have examined the effects of the muscarinic agonists, carbachol (CCh) and oxotremorine (Oxo), on the intracellular free Ca2+ concentration ([Ca2+]i) in acutely dissociated sympathetic neurons from adult rats using fura 2-based microfluorometry. The drugs increased [Ca2+]i by 86 +/- 7 and 38 +/- 10 nM for CCh and Oxo, respectively (both 10 microM). Basal [Ca2+]i was 52 +/- 3 nM. Depletion of the caffeine-sensitive Ca2+ store or blockade of the Ca(2+)-adenosinetriphosphatase with thapsigargin did not alter the effect of either agonist on the rise in [Ca2+]i. On the other hand, the omission of Ca2+ from the perfusion solution or the use of TA-3090, a Ca2+ channel antagonist, blocked the effects of CCh and Oxo. In whole cell current-clamp recordings, the muscarinic agonists elicited a depolarization and action potential firing, which probably explained the rise in [Ca2+]i observed with microfluorimetric recording. In addition to their direct effects on the [Ca2+]i, muscarinic agonists also reduced the rise in [Ca2+]i induced by a nicotinic agonist. This inhibitory effect, observed in 68% of cells that responded to the nicotinic agonist, was blocked by atropine and pertussis toxin, whereas the muscarinic agonist-induced increase in [Ca2+]i was blocked by atropine but was pertussis toxin insensitive. These results suggest that at least two muscarinic receptors are present on sympathetic neurons and that they mediate opposite effect on the fluctuation of [Ca2+]i.

Effects of Buthus martensii Karsch scorpion venom on the release of noradrenaline from in vitro and in vivo rat preparations.

The aim of this study was to test the neuronal effects of the Chinese Buthus martensii Karsch (BMK) scorpion venom in vivo and in vitro in order to understand the mechanism involved in the cardiovascular pressor effect of this venom. In conscious unrestrained rats, administration of 100 micrograms/kg i.v. BMK venom induced an increase in blood pressure, which was associated with a significant increase in plasma noradrenaline. In isolated atria, BMK also induced an increase in the stimulation-induced release of [3H]noradrenaline in a dose-dependent manner. The modulatory effect of agents acting at sympathetic prejunctional adrenoceptors on [3H]noradrenaline release was not altered by BMK venom administration. Finally, it was observed that 100 micrograms/mL BMK venom increased the intracellular calcium concentration in acutely dissociated sympathetic neurons from adult rat superior cervical ganglion. This action appeared to be mainly due to an influx of extracellular calcium. BMK venom induced a small rise in intracellular calcium in the absence of external calcium, indicating that it may also mobilize calcium from intracellular stores. The results observed in this study suggest that BMK venom may induce pressor responses by releasing noradrenaline from the sympathetic nerve terminals and that activation of neuronal calcium channels may be involved in that process.

Cardiovascular effects of Buthus martensii (Karsch) scorpion venom.

Buthus martensii (Karsch) (BMK) scorpion envenomation is a common medical problem in China and BMK scorpion has been widely used in traditional Chinese medicine. However, the cardiovascular effects of this venom have not been systematically investigated. In the present study, i.v. BMK venom injection significantly increased the blood pressure in conscious rats in a concentration-dependent manner (ED50 = 59 +/- 12 micrograms/kg). The increase in blood pressure occurred within 1 min of injection of the venom and was sustained for more than 50 min. Heart rate was not changed by the venom in conscious rats. In vitro studies with BMK venom revealed the increase in the force of contraction, without modification of the contraction frequency (within 20 min) of isolated atrial strips. Contractions of isolated arterial strips from aorta, renal and vertebral arteries were also enhanced by BMK venom with a time lag of 8 min between the application of the venom and the initiation of the contraction. Furthermore, BMK venom-induced rises in blood pressure in vivo and increased contraction of isolated vessel strips were inhibited by prazosin and tolazoline, respectively, two alpha 1-adrenergic antagonists. BMK venom alone did not alter intracellular calcium concentrations, [Ca2+]i, in cultured vascular smooth muscle cells and endothelial cells. However, BMK venom significantly increased the metabolism of InsP3 in dispersed cardiac myocytes, indicating a direct effect on cardiac myocytes. These results demonstrate the significant cardiovascular effects of BMK venom, which may be mediated by an alteration in InsP3 in cardiac myocytes but not by [Ca2+]i in vascular smooth muscle cells.

Neuropeptide Y and pancreatic polypeptide reduce calcium currents in acutely dissociated neurons from adult rat superior cervical ganglia.

We examined the effects of neuropeptide Y (NPY) and pancreatic polypeptide on calcium currents (ICa) in acutely dissociated neurons from the adult rat superior cervical ganglion. We found that NPY inhibited the ICa with an estimated IC50 value of 140 nM. This inhibitory effect appeared to be restricted to a subset of cells which were smaller in diameter than the general population. The effect of NPY on the ICa was prevented by pretreatment with pertussis toxin, suggesting the involvement of a GTP-binding protein of the Gi or Go subtype. omega-conotoxin GVIA also occluded the effects of NPY, which suggests that these were directed toward N-type Ca++ channels. The effects of NPY were mimicked by the fragment NPY (13-36) but not by peptide YY, indicating that a receptor distinct from a Y1- or a Y2-like NPY receptor was involved. Finally, we also observed that pancreatic polypeptide inhibited the ICa, suggesting that a pancreatic polypeptide receptor is also present on superior cervical ganglion neurons.

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.

Block of neuronal Ca++ influx by the anti-ischemic agent TA3090.

We examined the effect of the novel anti-ischemic drug, TA3090 on Ca++ entry into peripheral and central neurons. TA3090 inhibited voltage-dependent Ca++ entry into rat dorsal root ganglion (DRG) and sympathetic neurons. The degree of inhibition produced by the drug was voltage-dependent. TA3090 also inhibited Ca++ entry into DRG cells elicited by trains of action potentials. TA3090 inhibited Ca++ influx into neurons from the hippocampus. As in peripheral neurons, TA3090 blocked Ca++ influx associated with a Ca++ current in voltage-clamped cells or elicited by trains of action potentials. Furthermore, TA3090 blocked Ca++ influx stimulated by glutamate or N-methyl-D-aspartate. In addition, TA3090 blocked excitatory glutamate-mediated synaptic transmission between hippocampal pyramidal neurons. These data indicate that the protective effects of TA3090 in the brain under ischemic conditions may be partly or completely due to its ability to inhibit neuronal Ca++ influx.

Evidence that angiotensin II enhances noradrenaline release from sympathetic nerves in mouse atria by activating protein kinase C.

1. Mouse atria were incubated with [3H]-noradrenaline and the outflow of radioactivity induced by electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (1 x 10(-8) M) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001-1.0 x 10(-6) M) and phorbol 12, 13-dibutyrate (0.001-1.0 x 10(-6) M), protein kinase C activating phorbol esters, significantly enhanced the S-I outflow of radioactivity. Phorbol dibutyrate produced a greater maximal enhancement of S-I outflow of radioactivity than phorbol myristate acetate. The enhancement of S-I outflow of radioactivity produced by the combination of phorbol dibutyrate (1.0 x 10(-7) M) and phorbol myristate acetate (1.0 x 10(-7) M) was no greater than that produced by phorbol dibutyrate (1.0 x 10(-7) M) alone. The enhancement of S-I outflow of radioactivity produced by phorbol myristate acetate (1.0 x 10(-7) M) was constant whether the tissue was exposed for 15, 45 or 75 min. 3. When angiotensin II (1.0 x 10(-8) M) was present with the maximally effective concentration of phorbol dibutyrate (1.0 x 10(-7) M) it did not increase S-I outflow of radioactivity. 8-bromo-cyclic AMP (9.0 x 10(-5) M) by itself increased the S-I outflow of radioactivity and in the presence of the maximally effective concentration of phorbol dibutyrate the enhancement of S-I outflow of radioactivity produced by 8-bromo-cyclic AMP was maintained. 4. A protein kinase inhibitor, K-252a (1.0 x 10(-6) M), did not affect S-I outflow of radioactivity. K-252a significantly reduced the enhancement of S-I outflow of radioactivity produced by both phorbol myristate acetate (0.03 or 0.1 x 10(-6) M) and phorbol dibutyrate (0.01 or 1.0 x 10(-6) M). 5. K-252a (1.0 x 10(-6) M) blocked the enhancement of S-I outflow of radioactivity produced by angiotensin II (1.0 x 10(-8) M) and tetraethylammonium (1.0 x 10(-4) M). 6. These results suggest that angiotensin II receptors may enhance noradrenaline release through the pool of protein kinase C that is activated by phorbol dibutyrate.

Modulation by beta-adrenoceptors and angiotensin II receptors of splanchnic nerve evoked catecholamine release from the adrenal medulla.

In the present study, we have evaluated the effect of both facilitatory beta 2-adrenoceptor and angiotensin II receptor on the release of adrenal catecholamines induced by electrical stimulation of the splanchnic nerve in anaesthetized and vagotomized dog. In these experiments, individual or combined treatments with the beta 2-adrenoceptor antagonist ICI 118551 (0.3 mg/kg i.v.), the converting enzyme inhibitor captopril (2 mg/kg i.v.), or the angiotensin II receptor antagonist saralasin (2 micrograms.kg-1.min-1 i.v.) were found to significantly decrease the release of adrenal catecholamines during splanchnic nerve stimulation (5-V pulses of 2 ms duration for 3 min at 1 Hz) whatever the order of administration of the drugs. On the other hand, the infusion of angiotensin II (20 ng.kg-1.min-1) was shown to potentiate the release of adrenal catecholamines in response to electrical stimulation, and this effect was totally blocked by treatment with saralasin (4 micrograms.kg-1.min-1 i.v.). This facilitating angiotensin mechanism differed from beta-adrenoceptor facilitating mechanism, since following beta-blockade with ICI 118551, angiotensin II infusion still significantly potentiated the release of catecholamines during splanchnic nerve stimulation. These observations thus suggest that both facilitating beta 2-adrenoceptors and angiotensin II receptors can independently modulate the release of adrenal catecholamines.