L-3,4-dihydroxyphenylalanine-induced c-Fos expression in the CNS under inhibition of central aromatic L-amino acid decarboxylase.

L-3,4-dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic L-amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.

Neurobiology of L-DOPAergic systems.

L-DOPA is proposed to be a neurotransmitter and/or neuromodulator in CNS. It is released probably from neurons, which may contain L-DOPA as an end-product, and/or from some compartment other than catecholamine-containing vesicles. The L-DOPA itself produces presynaptic and postsynaptic responses. All are stereoselective and most are antagonized by competitive antagonist. In striatum, L-DOPA is neuromodulator, mother of catecholamines, not only a precursor for dopamine but also a potentiator of children for presynaptic beta-adrenoceptors to facilitate dopamine release and postsynaptic D2 receptors, and ACh release inhibitor. All may cooperate for Parkinson's disease. Meanwhile, supersensitization of increase in L-glutamate release to nanomolar levodopa was seen in Parkinson's model rats, which may relate to dyskinesia or "on-off" during chronic therapy. In lower brainstem, L-DOPA tonically activates postsynaptic depressor sites of NTS and CVLM and pressor sites of RVLM. L-DOPA is probably a neurotransmitter of primary baroreceptor afferents terminating in NTS. GABA, the inhibitory neuromodulator for baroreflex in NTS, tonically functions to inhibit, via GABAA receptors, L-DOPA release and depressor responses to levodopa. Levodopa inversely releases GABA. L-DOPAergic monosynaptic relay from NTS to CVLM and from PHN to RVLM is suggested. Tonic L-DOPAergic baroreceptor-aortic nerve-NTS-CVLM relay seems to carry baroreflex information. Disturbance of neuronal activity to release L-DOPA in NTS, loss of the activity in CVLM, enhancement of the activity with decreased decarboxylation and increase in sensitivity to levodopa in RVLM may be involved in maintenance of hypertension in SHR. This is a story of "L-DOPAergic receptors" with extremely high affinity and low density.

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA.

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.

Is L-dopa an endogenous neurotransmitter?

Since the 1960s, L-3,4-dihydroxyphenylalanine (L-dopa), a precursor of dopamine, has been thought to occur in the cytoplasm of catecholaminergic neurones. L-Dopa is traditionally believed to be an inert amino acid that exerts actions and effectiveness in Parkinson's disease via its conversion to dopamine by L-aromatic amino acid decarboxylase. In contrast to this generally accepted idea, Yoshimi Misu and Yoshio Goshima propose, in this Viewpoint article, that L-dopa itself is an endogenous neurotransmitter or neuromodulator in the CNS. This hypothesis is mainly based on the findings that L-dopa is released in a transmitter-like manner and that exogenously applied levodopa produces some responses.

Delta opioid receptor mediates phospholipase C activation via Gi in Xenopus oocytes.

Cloned mouse delta-subtype opioid receptor (DOR1) was expressed in Xenopus oocytes to study the signal transduction. Opioid delta-agonists evoked a calcium-dependent chloride current in oocytes injected with mRNA derived from DOR1, together with that from the alpha subunit of Gi1. The delta-agonist-induced current was blocked by naltrindol, a delta-specific antagonist. The delta-agonist evoked no or very weak currents in oocytes with the alpha subunit of Gq or G(o). These findings indicate the functional coupling between the opioid delta-receptor and phospholipase C through an activation of Gi.

Evidence for a metabostatic opioid kappa-receptor inhibiting pertussis toxin-sensitive metabotropic glutamate receptor-currents in Xenopus oocytes.

Glutamate evoked pertussis toxin-sensitive currents in Xenopus oocytes expressing metabotropic glutamate receptor subtype 1 (mGluR1) and exogenous Gi1 alpha. The mGluR1-currents were completely blocked by U-73122, a phospholipase C (PLC) inhibitor and by niflumic acid, a chloride channel blocker. In the oocyte further coinjected with poly(A)+ RNA from the guinea pig cerebellum, the mGluR1-currents were inhibited by U-50488H, an opioid kappa-agonist, and this inhibition was blocked by norbinaltorphimine, an opioid kappa-antagonist. These findings suggest that the mRNA encoding a novel subtype of opioid kappa-receptor which inhibits Gi1-PLC-mediated currents is present in guinea pig cerebellar poly(A)+ fractions.

The nucleus reticularis dorsalis: a region sensitive to physostigmine.

Microinjection of physostigmine (3 micrograms) into the nucleus reticularis dorsalis of the rat produced hypertension. Little or no increases in blood pressure were found at the other areas of the medullary reticular formation. The pressor effect following injection of physostigmine into the nucleus reticularis dorsalis was sensitive to atropine (1 microgram) but resistant to hexamethonium (5 micrograms), similarly injected. Atropine (1 microgram) bilaterally injected into the nucleus decreased pressor responses to intravenous physostigmine (100 micrograms/kg). These data may indicate that there are muscarinic receptors responsible for pressor effects in the nucleus reticularis dorsalis. The medullary cholinoceptors may be involved in the systemic physostigmine effect on blood pressure.

Altered tonic L-3,4-dihydroxyphenylalanine systems in the nucleus tractus solitarii and the rostral ventrolateral medulla of spontaneously hypertensive rats.

We have proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the central nervous system [Y. Misu et al. (1995) Adv. Pharmac. 32, 427-459]. L-DOPA as a probable neurotransmitter for the primary baroreceptor afferents tonically functions to mediate cardiodepressor control in the nucleus tractus solitarii and also tonically functions to mediate cardiopressor control in the rostral ventrolateral medulla of rats. We further attempted to clarify whether a transmitter-like L-DOPA system is altered in these areas of adult spontaneously hypertensive rats. By microdialysis in the left nucleus tractus solitarii area, the basal L-DOPA release was lower in spontaneously hypertensive rats than that in Wistar-Kyoto rats. This release was partially reduced by tetrodotoxin (1 microM) to the same absolute levels in the two strains. Tonic neuronal L-DOPA release is impaired in this nucleus of spontaneously hypertensive rats. This impairment is not secondarily due to decrease in formation or increase in decarboxylation of L-DOPA, since tyrosine hydroxylase activity was increased in spontaneously hypertensive rats, compared to Wistar-Kyoto rats, while no difference of L-aromatic amino acid decarboxylase activity was seen in the caudal dorsomedial medulla including the nucleus. L-DOPA (10-300 ng) microinjected into the nucleus produced dose-dependent hypotension and bradycardia. A maximum depressor response of spontaneously hypertensive rats to L-DOPA at higher doses was slightly greater than that of Wistar-Kyoto rats. On the other hand, in the left rostral ventrolateral medulla, the basal L-DOPA release was higher in spontaneously hypertensive rats than that in Wistar-Kyoto rats. This release was also partially reduced by tetrodotoxin to the same absolute levels in the two strains. Tonic neuronal L-DOPA release is enhanced in spontaneously hypertensive rats. This enhancement seems to include partially a decrease in decarboxylation of L-DOPA, since L-aromatic amino acid decarboxylase activity was decreased in spontaneously hypertensive rats compared to Wistar-Kyoto rats, while no difference in tyrosine hydroxylase activity was seen. L-DOPA (10-600 ng) produced dose-dependent hypertension and tachycardia. Importantly, a pressor response of spontaneously hypertensive rats to L-DOPA at lower doses was slightly greater than that of Wistar-Kyoto rats. L-DOPA seems to play a transmitter-like role in blood pressure regulation at levels of the nucleus tractus solitarii and rostral ventrolateral medulla in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

An L-dopaergic relay from the posterior hypothalamic nucleus to the rostral ventrolateral medulla and its cardiovascular function in anesthetized rats.

We have proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the central nervous system [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. Herein, we attempt to clarify whether lesions in the posterior hypothalamic nucleus decrease the tissue content of L-DOPA in the rostral ventrolateral medulla. We also attempt to clarify whether or not endogenous L-DOPA is evoked by electrical stimulation of the posterior hypothalamic nucleus. It is possible that evoked L-DOPA functions as a transmitter candidate to activate pressor sites of the rostral ventrolateral medulla in anesthetized rats. Electrolytic lesions were made in the bilateral posterior hypothalamic nucleus by a monopolar direct current of 2 mA for 10 s, 10 days before measurements. The effect of the lesions was to selectively decrease the tissue content of L-DOPA by one-half in the right rostral ventrolateral medulla. Decreases in the amounts of dopamine, noradrenaline or adrenaline were not observed. Decreases were also not evident in the right caudal ventrolateral medulla. During microdialysis of the right rostral ventrolateral medulla, extracellular basal levels of L-DOPA and three types of catecholamine were consistently detectable by high-performance liquid chromatography with electrochemical detection. Tetrodotoxin (1 microM) perfused into the right rostral ventrolateral medulla gradually decreased basal levels of L-DOPA by 25%; it decreased basal levels of noradrenaline and adrenaline by 25-30% and dopamine levels by 40%. Intensive electrical stimulation of the ipsilateral posterior hypothalamic nucleus (50 Hz, 0.3 mA, 0.1 ms duration, twice for 5 min at an interval of 5 min) selectively caused the release of L-DOPA in a repetitive and constant manner. The stimulation was accompanied by hypertension and tachycardia. However, catecholamines were not released. Tetrodotoxin suppressed the release of L-DOPA, but partially inhibited hypertension with only a slight inhibition of tachycardia evoked by stimulation of the posterior hypothalamic nucleus. L-DOPA methyl ester, a competitive L-DOPA antagonist, was bilaterally microinjected into pressor sites of the rostral ventrolateral medulla at 1.5 microg x 2 and 3 microg x 2. The antagonist dose-dependently and consistently antagonized pressor and tachycardiac responses to mild transient stimulation of the unilateral posterior hypothalamic nucleus (33 Hz, 0.2 mA, 0.1 ms duration, for 10 s). In addition, the antagonist alone (3 microg x 2) elicited hypotension and bradycardia. These results show that an L-DOPAergic relay may project from the posterior hypothalamic nucleus directly to pressor sites of the rostral ventrolateral medulla and/or indirectly to certain neurons near pressor sites in microcircuits of the same region. When released, L-DOPA appears to function tonically to activate pressor sites; it also appears to be involved in the maintenance and regulation of blood pressure and heart rate.

L-dopaergic components in the caudal ventrolateral medulla in baroreflex neurotransmission.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is probably a transmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii; L-DOPA functions tonically to activate depressor sites of the caudal ventrolateral medulla, which receives input from the nucleus tractus solitarii [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. We have attempted to clarify whether or not L-DOPAergic components within the caudal ventrolateral medulla are involved in baroreflex neurotransmission in anesthetized rats. Electrolytic lesions of the right nucleus tractus solitarii (1 mA d.c. for 10 s, 10 days before measurement) selectively decreased by 45% the tissue content of L-DOPA in the dissected ipsilateral caudal ventrolateral medulla. Electrolytic lesions did not decrease dopamine, norepinephrine and epinephrine levels. During microdialysis of the right caudal ventrolateral medulla, extracellular levels of L-DOPA, norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid were consistently detectable using high-performance liquid chromatography with electrochemical detection. However, extracellular dopamine levels were lower than the assay limit. Baroreceptor activation by i.v. phenylephrine selectively evoked L-DOPA without increasing the levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. This L-DOPA release was suppressed by acute lesion in the ipsilateral nucleus tractus solitarii. Intermittent stimulation of the right aortic depressor nerve (20 Hz, 3 V, 0.3 ms duration, for 30 min) repetitively and constantly caused L-DOPA release, hypotension and bradycardia, without increases in levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. Local inhibition of L-DOPA synthesis with alpha-methyl-p-tyrosine (30 microM) infused into the ipsilateral caudal ventrolateral medulla gradually decreased basal levels of L-DOPA and 3,4-dihydroxyphenylacetic acid without decreasing norepinephrine and epinephrine. The inhibition of L-DOPA synthesis interrupted L-DOPA release and decreased by 65% depressor responses elicited by aortic nerve stimulation; however, it produced no effect on bradycardic responses. CoCl2 (119 ng), a mainly presynaptic inhibitory transmission marker, and L-DOPA methyl ester (1 microg), a competitive L-DOPA antagonist, when microinjected into depressor sites of the right caudal ventrolateral medulla, reduced by 60% depressor responses to transient ipsilateral stimulation of the aortic nerve (20 Hz, 3 V, 0.1 ms duration, for 10 s). No changes in bradycardic responses were observed. There may exist an L-DOPAergic relay from the nucleus tractus solitarii to the caudal ventrolateral medulla. L-DOPAergic components in the caudal ventrolateral medulla are involved in baroreflex neurotransmission via a baroreceptor-aortic depressor nerve-nucleus tractus solitarii-caudal ventrolateral medulla relay in the rat.

Baroreceptor-aortic nerve-mediated release of endogenous L-3,4-dihydroxyphenylalanine and its tonic depressor function in the nucleus tractus solitarii of rats.

We have proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter and/or neuromodulator in the central nervous system [Misu Y. and Goshima Y. (1993) Trends pharmac. Sci. 14, 119-123]. This study aimed to explore whether or not endogenous L-DOPA, as a neurotransmitter candidate of the primary baroreceptor afferents, tonically functions to activate depressor neurons in the nucleus tractus solitarii of anesthetized rats. By parallel microdialysis in bilateral nucleus tractus solitarii areas, the basal L-DOPA release was in part inhibited by tetrodotoxin perfusion (1 microM) or Ca2+ deprivation, and was markedly reduced by alpha-methyl-p-tyrosine (200 mg/kg, i.p.), a tyrosine hydroxylase inhibitor. Forty to 100 mM K+ concentration-dependently released L-DOPA. Fifty millimoles K+ repetitively and constantly released L-DOPA. This release was Ca(2+)-dependent. Stimulation of the left aortic nerve (100 Hz, 8 V) repetitively and constantly released L-DOPA and this release was tetrodotoxin-sensitive. Phenylephrine i.v. infused produced L-DOPA release and reflex bradycardia, temporally associated with a rise and subsequent recovery of blood pressure. This release and bradycardia were abolished by denervation of the bilateral carotid sinus and aortic nerves. In addition, L-DOPA methyl ester, a competitive L-DOPA antagonist, when microinjected into depressor sites of the left nucleus tractus solitarii, antagonized depressor responses to mild stimulation (20 Hz, 3 V) of the ipsilateral aortic nerve. This antagonist alone, microinjected bilaterally, elicited a dose-dependent hypertension, which was abolished by alpha-methyl-p-tyrosine. Furthermore, by immunocytochemical analysis seven days after denervation of the left aortic nerve, tyrosine hydroxylase- and L-DOPA-, but not dopamine- and dopamine-beta-hydroxylase-immunoreactivities decreased in the ipsilateral nucleus tractus solitarii and dorsal motor vagus nucleus complex area. In the left ganglion nodosum, denervation decreased staining and number of L-DOPA-immunoreactive cells and staining of tyrosine hydroxylase-immunoreactive cells, but no modification of dopamine-immunoreactive cells was seen. Taken together with previous findings that L-DOPA itself is stereoselectively responsible for cardiovascular control in this nucleus, it is probable that L-DOPA is a neurotransmitter of the primary baroreceptor afferents terminating directly in depressor neurons and/or indirectly in some neurons within a microcircuit, including depressor neurons of the nucleus tractus solitarii. Endogenously released L-DOPA itself tonically functions to activate depressor neurons for regulation of blood pressure in the rat nucleus tractus solitarii.

Autoradiographic studies using L-[(14)C]DOPA and L-DOPA reveal regional Na(+)-dependent uptake of the neurotransmitter candidate L-DOPA in the CNS.

We previously proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the CNS. Receptor and transporter molecules for L-DOPA, however, have not been determined. In the present study, in order to localize the uptake sites of L-DOPA in the CNS, we performed autoradiographic uptake studies using L-[14C]DOPA and L-[3H]DOPA in the uptake study on rat brain slice preparations, and further analyzed the properties of L-DOPA uptake. Image analysis of the L-[14C]DOPA autoradiogram showed a unique heterogeneous distribution of uptake sites in the brain. The intensity was relatively high in the cerebral cortex, the hypothalamus, the cerebellum and the hippocampus, while the density was moderate or even low in the striatum and the substantia nigra. L-DOPA and phenylalanine, but not dopamine (10mM) were able to almost completely inhibit the uptake of L-[14C]DOPA to basal levels. Microautoradiographic studies using L-[3H]DOPA revealed accumulation of dense grains in the median eminence, the supraoptic nucleus of the hypothalamus, the cerebral cortex (layer I) and the hippocampus. In the cerebellum, grains formed in clusters surrounding the Purkinje cells. This grain accumulation was concluded to be in Bergmann glial cells, since the morphological pattern of grain accumulation was similar to that of the immunoreactivity of the glutamate aspartate transporter, a marker protein for Bergmann glial cells. In the hippocampus, the grain density significantly decreased under Na(+)-free conditions. In addition, grain density also decreased in the absence of Cl(-). In contrast, grains in the choroid plexus and the ependymal cell layer, were not affected by the absence of Na(+). These findings indicated that the uptake of L-DOPA occurs via various types of large neutral amino acid transport mechanisms. It appears that neuronal and/or glial cells, which take up L-DOPA in a Na(+)-dependent manner, exist in the CNS. Our finding further supports the concept that L-DOPA itself may act as a neurotransmitter or neuromodulator.

Prevention by guanethidine analogues of output of noradrenaline induced by sodium reduction in rabbit ventricular slices.

1 The prevention by guanethidine and related agents of the output of noradrenaline induced by low sodium was investigated in rabbit ventricular slices. When external NaCl was reduced, the output of noradrenaline into the medium collected at 30 min intervals, increased and the endogenous levels decreased. These changes induced by replacing sodium with sucrose or choline were not affected either by the omission of calcium and addition of 0.5 mM ethylene glycol-bis(aminoethylether)N,N,N',N' tetra-acetic acid (EGTA) or by an increase in the calcium concentration to 10 mM 30 min before sodium deprivation.2 Guanethidine 4 x 10(-6) and 4 x 10(-5) M and 4-7-exo-methylene-hexahydroisoindoline-ethyl guanidine (No. 865-123) 4 x 10(-5) to 8 x 10(-4) M inhibited, in a dose-dependent manner, increases in output of noradrenaline induced by reduction of sodium to 18 mM, while guanethidine 8 x 10(-5) M and high doses of bretylium produced no inhibition: the latter two released noradrenaline.3 The inhibitory actions of guanethidine 4 x 10(-5) M and No. 865-123 4 x 10(-4) M were prevented by tetracaine 3.3 x 10(-4) M, which per se did not modify the output of noradrenaline induced by 18 mM sodium.4 Accumulation of guanethidine and No. 865-123 in ventricular slices was greater than that noted in striated muscle slices and was dose-, time- and temperature-dependent. Tetracaine 3.3 x 10(-4) M did not prevent the accumulation of guanethidine 4 x 10(-5) M and No. 865-123 1.1 x 10(-6) to 4 x 10(-4) M.5 The guanidine derivatives appear to increase the permeability of adrenergic nerve endings to sodium ions.

Further evidence for the prevention by guanethidine of noradrenaline efflux from rabbit ventricular slices induced by a low external sodium concentration.

When guanethidine was applied to rabbit ventricular slices at 37 degrees C for an initial 30 min incubation period, doses of 4 X 10(-5) and 8 X 10(-5) M irreversibly inhibited increases in efflux of noradrenaline induced by the subsequent reduction of external sodium concentrations to 18 mM. This inhibition was prevented if the temperature was reduced to 0 degrees C during the drug application period. Guanethidine transported into adrenergic nerve endings appears to increase permeability to Na+, thereby leading to the reduction in efflux.

Action of guanethidine on rabbit atrial membranes.

1 Intracellular potentials were recorded in driven left atria from reserpine-treated rabbits. Guanethidine 2 X 10(-5) M slightly increased Vmax and shortened the total duration (TD) of the action potential (AP) without causing hyperpolarization. For the first 30 min after 4 X 10(-4) M, Vmax increased without hyperpolarization and AP height increased slightly. Thereafter, Vmax and height decreased with a slight and gradual depolarization. This depolarization was irreversible. TD was increased after 15 minutes. Guanethidine 2 X 10(-3) M initially decreased Vmax and height before causing depolarization. 2. Pretreatment with tetrodotoxin (TTX) 1.6 X 10(-7) M prevented or reversed the initial increases in Vmax, height and TD induced by guanethidine (4 X 10(-4) M). 3 TTX 3.1 to 6.2 X 10(-6) M, added 15 or 30 min after guanethidine 4 X 10(-4) M, delayed or prevented depolarization by guanethidine. 4 Ouabain 10(-5) M incubated for 20 and 90 min greatly inhibited Na+, K+-adenosine triphosphatase and K+-phosphatase activities; guanethidine was without effect. 5 Guanethidine probably increases resting sodium permeability after the promotion of increases in sodium permeability during the AP. High doses of the drug decrease sodium permeability during the AP.

Comparative studies of (--)-, (+/-)-propranolol, atenolol, guanethidine, bretylium and tetracaine on adrenergic transmission.

1 Effects of (--)-, (+/-)-, and (+)-propranolol, atenolol, guanethidine, bretylium and tetracaine were studied on relaxation responses of rabbit ileum and contractile responses of rabbit pulmonary artery and guinea-pig vas deferens to electrical nerve stimulation (2 to 50 Hz). 2 In the ileum, inhibition by tetracaine 3.3 x 10(-6) M occurred at high frequencies of stimulation, while bretylium 1.2 x 10(-4) M and guanethidine 2 x 10(-5) M inhibited responses at all frequencies, the latter producing greater inhibition at low frequencies. 3 (+/-)-Propranolol 10(-5) M produced a tetracaine-type inhibition after 1 h and a bretylium-pattern after 2 h in the ilea and pulmonary arteries and a transition from bretylium- to guanethidine-pattern in the vas deferens, while atenolol 2 x 10(-5) to 10(-4) M produced guanethidine-type inhibition in all preparations. 4 (--)-, (+/-)-, and (+)-Propranolol 3 x 10(-6) to 3.3 x 10(-5) M were equipotent in the vas deferens and ileum. However, inhibition by (--)-propranolol 3.3 x 10(-5) M persisted in the ileum, while that by the (+)-isomer was partially restored by washing. 5 (--)- or (+)-Propranolol 3.3 x 10(-5) M or atenolol 2 x 10(-5) M did not inhibit relaxation of the ileum after the bath temperature was maintained at 4 degrees C for 2 h during drug application. 6 In conclusion, propranolol and atenolol both have gradually developing guanethidine-like adrenergic neurone blocking actions.

Biphasic actions of L-DOPA on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices.

Effects of L-DOPA on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices evoked by electrical field stimulation at 5 Hz were investigated in the absence and presence of p-bromobenzyloxyamine (NSD-1055), a DOPA-decarboxylase inhibitor. In the absence of NSD-1055, L-DOPA produced a facilitation of impulse-evoked release of noradrenaline at 0.1 microM but not at 1 and 10 microM, and had no effect on the spontaneous release. On the other hand, L-DOPA 0.1 to 10 microM dose-dependently increased the spontaneous release of dopamine and the highest concentration only increased the evoked release and tissue content of dopamine. In the presence of NSD-1055 10 microM, the increase in the spontaneous release of dopamine was prevented and L-DOPA produced biphasic regulatory effects on the evoked release of noradrenaline and dopamine, a facilitation at 0.1 microM and an inhibition at 1 microM. The facilitation was antagonized by (-)-propranolol 0.1 microM, but not by the (+)-isomer, whereas the inhibition was antagonized by S-sulpiride 1 nM, but not by the R-isomer. In conclusion, L-DOPA appears to produce biphasic actions on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices, not through its conversion to dopamine but through presynaptic regulatory mechanisms, an inhibition via dopamine receptors at a micromolar concentration and a facilitation via beta-adrenoceptors at the lower concentration.

Evidence for tonic activation of prejunctional beta-adrenoceptors in guinea-pig pulmonary arteries by adrenaline derived from the adrenal medulla.

1. The effects of (+/-)-carteolol 10(-8) M to 10(-6) M, a non-selective beta-antagonist, applied cumulatively, on stimulation-evoked 3H-release at 1 Hz were studied in pulmonary arteries isolated from guinea-pigs. The guinea-pigs were subjected to either bilateral adrenalectomy, adrenalectomy followed by injections of deoxycorticosterone acetate (DOCA) and hydrocortisone, bilateral adrenodemedullation or a sham operation, and then loaded in vitro with [3H]-noradrenaline. 2. Carteolol inhibited 3H-output in arteries from sham-operated animals in a concentration-dependent manner. This inhibitory effect was not found in pulmonary arteries from animals subjected to adrenalectomy or adrenodemedullation. However, DOCA and hydrocortisone pretreatment, did not prevent the disappearance of the carteolol-induced inhibition of 3H-release. 3. Adrenalectomy and adrenodemedullation depleted or markedly reduced the endogenous contents of adrenaline in pulmonary arteries without altering the levels of dopamine and noradrenaline. 4. It is concluded that adrenaline, mainly derived from the adrenal medulla, acts as an endogenous agonist for tonically functioning prejunctional beta-adrenoceptors in guinea-pig pulmonary arteries, probably by being taken up and co-released with noradrenaline.

Protein kinase C inhibitor potentiates the agonist-induced GTPase activity in COS cell membranes expressing delta-opioid receptor.

In COS-7 cell membranes expressing cloned delta-opioid receptor, [D-Ser2, Leu5]enkephalin-Thr6, an opioid delta-agonist, showed no significant stimulation of high-affinity GTPase, while this agonist binding showed a guanine nucleotide sensitivity. Significant stimulation of GTPase activity by this agonist was observed only when the cells were pretreated with 0.1 microM calphostin C, a protein kinase C inhibitor, and when this inhibitor was further added to the reaction mixture at 1 microM. These findings suggest that protein kinase C is involved in the heterologous desensitization of delta-opioid receptor in the cells.

Opioid mu- and kappa-receptor mediate phospholipase C activation through Gi1 in Xenopus oocytes.

In the Xenopus oocytes expressing mu- or kappa-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 alpha RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the mu- and kappa-receptors mediate phospholipase C activation through Gi1 alpha, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte.