Upregulation of immunoreactive angiotensin II release and angiotensinogen mRNA expression by high-frequency preganglionic stimulation at the canine cardiac sympathetic ganglia.

The possible involvement of the local angiotensin system in ganglionic functions was investigated in the canine cardiac sympathetic ganglia. Positive chronotropic responses to preganglionic stellate stimulation at high frequencies, after intravenous administration of pentolinium plus atropine, were inhibited by the nonpeptide angiotensin AT(1) receptor antagonist forasartan or the angiotensin I-converting enzyme inhibitor captopril, whereas the rate increases elicited by the postganglionic stellate stimulation and norepinephrine given intravenously failed to be inhibited by these antagonists. The levels of endogenous immunoreactive angiotensin II, as determined by radioimmunoassay in the incubation medium of the stellate and inferior cervical ganglia, were increased after the high-frequency preganglionic stimulation of the isolated ganglia. The increment of the peptide was also antagonized by the pretreatment with captopril but not by a chymase inhibitor, chymostatin. The expression of angiotensinogen mRNA was observed in the stellate ganglion, adrenal, liver, and lung but not in the ovary and spleen. The expression of the mRNA in the stellate and inferior cervical ganglia increased after high-frequency preganglionic stimulation of the in vivo dogs for a period of 1 hour. These results indicate that an intrinsic angiotensin I-converting enzyme-dependent angiotensin system exists in the cardiac sympathetic ganglia, which is activated by high-frequency preganglionic stimulation.

Antineoplastic effects in rats of 5-fluorocytosine in combination with cytosine deaminase capsules.

5-Fluorocytosine (5-FC) lacks antineoplastic activity in human subjects because of the absence of cytosine deaminase (CDase) in mammalian cells. Intratumoral conversion of 5-FC into 5-fluorouracil (5-FUra) by locally implanted capsules containing CDase followed by systemic administration of 5-FC can be expected to induce antineoplastic activity at a local site with minimal systemic toxicity. In vitro and in vivo experiments were performed to evaluate this hypothesis. Spectrophotometric analysis confirmed the deamination of 5-FC to 5-FUra by CDase extracted from cultivated Escherichia coli. In vitro studies showed that 5-FC combined with CDase induced significant growth-inhibitory effects on the cultured glioma cells. An active CDase capsule, made of cellulose tubing, was newly designed for local implantation. 5-FC concentrations in the s.c. tumors of the rats given these CDase capsules, followed by 5-FC administration, showed a sufficient amount of delivery of 5-FC to the tumor tissue. 5-FUra appearing in the tumor reached the level of 8.0 micrograms/g at 2 h and stayed at more than 1.0 microgram/g at between 1 and 6 h. Significant reduction of the tumor growth and cytotoxic changes were observed. The passive cutaneous anaphylaxis reaction demonstrated no allergic reaction to the host due to the capsule. These results suggest that this chemotherapeutic method is effective for human brain tumors.

Release of purines and noradrenaline by ouabain and potassium chloride from vascular adrenergic nerves.

1 Release of [3H]-noradrenaline and 3H-purine by ouabain (10(-4)M) or high KCl (50 mM) was investigated in the superfused rabbit pulmonary arterial segment preincubated with [3H]-noradrenaline or [3H]-adenosine. 2 Ouabain elicited a delayed large contraction and a parallel [3H]-noradrenaline efflux. These were substantially inhibited by Ca2+-free medium or preincubation with 6-hydroxydopamine (30 micrograms/ml, 30 min). 3 Ouabain caused an 3H-purine efflux which was slower than the [3H]-noradrenaline efflux. This was inhibited by 6-hydroxydopamine and in part by phentolamine (3 X 10(-6)M), indicating both neuronal and extraneuronal origins of purines. 4 In contrast to ouabain, high KCl appeared to induce predominantly purine efflux, which was phentolamine-insensitive and 6-hydroxydopamine-sensitive, indicative of neuronal origin. 5 It is suggested that the purine efflux evoked by ouabain and high KCl may originate from different neuronal vesicles.

Facilitation by clonidine of purine release induced by high KCl from the rabbit pulmonary artery.

1 The effect of clonidine on the 3H-purine release evoked by KCl or (-)-adrenaline was assessed in the superfused helical strip of the rabbit pulmonary artery pretreated with [3H]-adenosine. 2 Clonidine (3 x 10(-5) M to 10(-4) M) significantly enhanced the 3H-purine efflux evoked by 50 mM KCl but not by 3 x 10(-6) M) (-)-adrenaline. 3 This facilitatory effect of clonidine on the KCl-induced purine release was unaltered by phentolamine 3 x 10(-6) M. It was absent in arterial segments denervated with 6-hydroxydopamine 30 microgram/ml. 4 A sustained contractile response was evoked by clonidine 3 x 10(-5) M without an increase in the 3H-purine efflux. This was significantly reduced by phentolamine 3 x 10(-6) M, but not by yohimbine 10(-5) M or by denervation with 6-hydroxydopamine. 5 The uptake of [3H]-adenosine into the segments was not inhibited by clonidine 3 x 10(-5) M. 6 It is suggested that the facilitation by clonidine of the KCl-induced purine release is due to prevention of presynaptic autoinhibition of purine release from adrenergic nerves, by an antiadenosine action of the drug.

Contribution of intra- and extracellular Ca2+ to noradrenaline exocytosis induced by ouabain and monensin from guinea-pig vas deferens.

1. Contributions of intra- and extracellular Ca2+ to noradrenaline (NA) release evoked by increasing intracellular Na+ concentrations (ouabain plus monensin) from adrenergic nerves of guinea-pig vas deferens were evaluated under conditions eliminating carrier-mediated NA release (with 100 microM cocaine). 2. Ouabain (100 microM) plus monensin (10 microM), unlike 100 mM KCl, produced a marked NA release which was unchanged by Ca(2+)-removal. 3. In normal solution but not in Ca(2+)-free solution, the release of NA evoked by ouabain plus monensin was reduced by adenosine, clonidine and neuropeptide Y, and by Ca(2+)-channel blockers such as omega-conotoxin GVIA and nifedipine. The release of NA was also decreased by cromakalim in a glibenclamide-sensitive fashion. 4. In contrast, in the absence but not in the presence of Ca2+, the drug-evoked NA release was inhibited by mitochondrial inhibitors (carbonylcyanide-m-chlorophenylhydrazone and oligomycin) and further by immobilizers of intracellular Ca2+ (TMB-8 and BAPTA-AM) and calmodulin antagonists (W-7 and trifluoperazine). 5. These findings suggest that the release of NA evoked by elevation of [Na+]i from adrenergic nerves in the presence and absence of Ca2+ involves, in part, exocytotic processes which are triggered by depolarization-induced Ca2+ influx and by utilization of Ca2+ from intracellular Ca2+ store sites such as mitochondria, respectively.

Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and alpha, beta-methylene ATP in guinea-pig urinary bladder.

1. The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea-pig urinary bladder and compared with those evoked by ATP and alpha, beta-methylene ATP (a P2-purinoceptor agonist). 2. AP4A, ATP and alpha, beta-methylene ATP produced concentration-dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate-6-azophenyl- 2'-4'-disulphonic acid), 0.3- 30 microM, a P2x-purinoceptor antagonist, and suramin, 1-300 microM, a P2-purinoceptor antagonist in a concentration-dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and alpha, beta-methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3. AP4A (10 microM) and ATP (100 microM) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of alpha, beta-methylene ATP (1 microM). 4. In fluorescence experiments with fura-2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L-type Ca2+ channel blocker. 5. These findings suggest that P2x-purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea-pig urinary bladder, and that the AP4A-evoked contraction results from Ca2+ influx.

Antagonism by nifedipine of contraction and Ca2(+)-influx evoked by ATP in guinea-pig urinary bladder.

1. The effects of Ca2(+)-antagonists, especially nifedipine, on contraction and increase of intracellular Ca2+ (Fura-2/AM method) evoked by ATP were evaluated in a thin outer layer segment of guinea-pig urinary bladder. 2. The ATP-evoked contraction was markedly inhibited by dihydropyridine-type Ca2(+)-antagonists, such as nifedipine and nitrendipine, but not by D-600, omega-conotoxin and tetramethrin. 3. This antagonism by nifedipine of ATP-evoked contractions was competitive from the Schild plot analysis, the pA2 value being 8.23. The reduction of ATP-evoked contraction by nifedipine (0.1 microM) was fully reversed by administration of Bay K 8644 (0.1 microM). 4. ATP (100 microM) caused an increase of fluorescence brightness after loading Fura-2/AM, which was coupled with a contraction of the bladder. Both the contraction and the elevation of intracellular Ca2+ evoked evoked by the nucleotide were completely antagonized by nifedipine. by the nucleotide were completely antagonized by nifedipine. 5. These results suggest that ATP may activate the dihydropyridine-sensitive, voltage-dependent Ca2(+)-channels in a direct or indirect fashion and, thereby, elicit a contraction of the bladder.

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle.

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by alpha, beta-methylene ATP (alpha, beta-mATP) and other P2-agonists in guinea-pig ileum. 2. alpha, beta-mATP 100 microM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED50 values were 5 and 25 microM, respectively. The maximum release of ATP by alpha, beta-mATP was markedly reduced by 250 microM suramin, 30 microM pyridoxal-phosphate-6-azophenyl-2',5'-disulphonic acid (PPADS) and 30 microM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2. 3. Although the contraction caused by alpha, beta-mATP was strongly diminished by Ca(2+)-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 microM, the release of ATP was virtually unaffected by these procedures. 4. UTP, beta, gamma-methylene ATP (beta, gamma-mATP) and ADP at 100 microM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment. 5. The potency order of all the agonists tested for the release of ATP was alpha, beta-mATP > UTP > beta, gamma-mATP > ADP. 6. In superfusion experiments with cultured smooth muscle cells from the ileum, alpha, beta-mATP (100 microM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2. 7. These findings suggest that ATP release and contraction induced by P2-agonists such as alpha, beta-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors. P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.

Involvement of adenosine receptor activities in aggressive responses produced by clonidine in mice.

A behavioral study was made of the mechanisms underlying the aggressive behavior induced by high doses of clonidine in mice. The frequency of clonidine-induced aggressive responses such as attacking and biting was increased dose-dependently from 10 to 50 mg/kg. Aggressive behavior induced by clonidine at doses of 10-30 mg/kg was potentiated under conditions of isolation and food deprivation for 24 h. Clonidine (30 mg/kg)-induced aggressive behavior was attenuated by adenosine (10 mg/kg IP) or dipyridamole (10 mg/kg IP), but markedly antagonized by combined pretreatment with both drugs. The behavior was strongly reduced by potent adenosine analogs, such as N6-cyclohexyl adenosine (CHA, 0.1 and 0.2 mg/kg IP) and N6-(L-phenyl isopropyl) adenosine (L-PIA, 0.2 mg/kg IP), but conversely was potentiated by phentolamine (10 mg/kg IP) or theophylline (10 mg/kg IP). Diazepam (2.5 mg/kg IP) and Ro15-1788 (2.5 mg/kg IP), a benzodiazepine receptor antagonist, also blocked the aggressive behavior. The inhibition by CHA (0.2 mg/kg IP) or diazepam (2.5 mg/kg) of clonidine-induced aggression was not antagonized by additional pretreatment with bicuculline (2 mg/kg IP). The aggressive response to apomorphine (8 mg/kg IP) was not affected by those drugs which inhibited the response to clonidine. The results suggest that the aggressive behavior evoked by high doses of clonidine, but not that by apomorphine, involves a blockade of adenosine receptors.

Muscarinic acetylcholine receptor-mediated increase of angiotensin type 2 receptor mRNA in PC12 cells.

The present study examined changes in the angiotensin type 2 (AT2) receptor mRNA level after carbachol exposure in PC12 cells. The AT2 receptor mRNA level increased 2- to 3-fold after 12-18 h of carbachol exposure (100 microM). The up-regulation of AT2 receptor mRNA was antagonized by atropine, which is a muscarinic receptor antagonist, thus suggesting that the increase in AT2 receptor mRNA is mediated via muscarinic acetylcholine receptors. This increase is blocked by NG-nitro-L-arginine-methylester, nitric oxide (NO) synthase inhibitor, and hemoglobin NO trap. Protein kinase C (PKC) inhibitors, H-7 and calphostin C, inhibited the carbachol-induced upregulation. In addition, a G kinase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), inhibited this increase. These findings suggest that the carbachol-induced increases in AT2 receptor mRNA is regulated by the activation of NO-cGMP and/or the PKC pathway.

Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells.

We describe an application of gel microdroplet (GMD) and flow cytometry techniques to selective enrichment of non-growing Leuconostoc mesenteroides cells, which are well culturable on other media, from a mixture with Bacillus subtilis cells in nutrient broth. After encapsulating cells of the mixed population within GMDs and a brief incubation in nutrient broth, the inability of L. mesenteroides cells to form microcolonies within GMDs allowed their discrimination from B. subtilis cells. After staining the GMD mixture with 6-carboxyfluorescein diacetate, which showed no influence on cell viability, the GMDs containing single cells of L. mesenteroides were selectively collected using flow cytometry sorting based on differences in fluorescence intensity. The cells of L. mesenteroides retained viability during the process.

cDNA cloning and expression of rat leukotriene C(4) synthase: elevated expression in rat basophilic leukemia-1 cells after treatment with retinoic acid.

Leukotriene C(4) synthase (LTC(4) S) is considered a pivotal enzyme for generation of potent proinflammatory mediators, cysteinyl-leukotrienes (cysLTs). LTC(4) S cDNA was cloned in rat basophilic leukemia-1 (RBL-1) cells, and exhibited 84.8% and 94.5% identity with the reported human and mouse LTC(4) S cDNA sequences, respectively. Homology between the rat LTC(4) S amino acid sequence and the corresponding sequences from the other species was 86.5% and 95.3% with human and mouse sequences, respectively. Rat LTC(4) S thus showed extensive homology with both mouse and human cDNA sequences. The active enzyme as assessed by LTC(4) S activity was expressed in COS-7 cells. While RBL-1 cells after the culture for 48 h in the presence of 0.1 microg/ml all trans -retinoic acid (RA) exhibited 27 times higher LTC(4) S activity than control cells, Northern-blot analysis of RA-treated cells showed upregulation of LTC(4) S mRNA. Polyclonal antibody was raised against the synthesized peptide deduced from the nucleotide sequence. Thus, Western-blot analysis of RBL-1 cells treated with RA and COS-7 cells transfected with pcDNA-LTC(4) S commonly showed a band at approximately 18 kDa in each solubilized enzyme solution, but either control cells did not. This cDNA probe and antibody may be useful for investigating the roles of cysLTs in various experimental models of rats.

Possible involvement of central C-type natriuretic polypeptide receptor on water intake in spontaneously hypertensive rats, but not in normotensive Wistar-Kyoto rats.

The present study was designed to examine the possible role of brain C-type natriuretic polypeptide receptor (GC-B) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). The level of GC-B mRNA in various regions of the brain in both SHR and WKY was examined in the present study. The GC-B mRNA was unevenly distributed in rat brain, the transcript being expressed predominantly in the hypothalamus and cerebellum but comparatively at low level in the striatum and septum. However, the level in the septum was 3-fold higher in SHR than than in age-matched WKY, while no differences were observed in other regions of the brain. Intracerebroventricular administration of antisense oligonucleotide to GC-B mRNA inhibits the night-time water intake in SHR, but not in WKY. However, the daily food intake was not significantly altered by the injection of antisense oligonucleotide in both strains. These results demonstrate that the brain GC-B mRNA, particularly in septum, is increased in SHR and this increase may be closely related to the regulation of water-drinking behaviour in SHR.

The increase in angiotensin type-2 receptor mRNA level by glutamate stimulation in cultured rat cortical cells.

The changes in the angiotensin type-2 (AT2) receptor mRNA level during glutamate neurotoxicity in cultured rat cortical cells are examined to assess the possible involvement of AT2 receptor in cell injury. The day 10-14 cortical neurons were exposed to glutamate at a toxic concentration of 100 microM for 15 min. The viability of the culture was reduced by 60% after 24 h. AT2 receptor mRNA was then increased 2-fold after exposure to glutamate, while the maximum increase was observed in a dose-dependent manner (50-1000 microM) 3 h after glutamate stimulation. AT2 receptor binding also increased 3-12 h after glutamate exposure. The results suggest that the increase in the AT2 receptor preceded to some extent the insult of the cell after exposure. The increase in the mRNA level was suppressed by MK-801, N-methyl-D-aspartate (NMDA) receptor antagonist, thus indicating the possible involvement of NMDA receptor. The increase in the mRNA level was also antagonized by N-nitro L-arginine methyl-ester, a nitric oxide synthase inhibitor. The hemoglobin, a nitric oxide trap, inhibited the increase in the mRNA level. These results suggest that the increase in the mRNA level is associated with the nitric oxide synthesis by glutamate exposure. The viability of cortical cells after glutamate stimulation was partially restored by the AT2 receptor antagonist and by the antisense oligonucleotide for the AT2 receptor. The present results thus suggest that the AT2 receptor may in some way be related to one of the processes in cell injury caused by glutamate.

Existence and pharmacological properties of dopamine D4 receptors in guinea pig vas deferens.

This study was carried out to identify subtypes of dopamine D2-like receptors in guinea pig isolated vas deferens. Dopamine had no effect on the muscle tone in the presence of prazosin, an alpha1-adrenoceptor antagonist. However, contractile responses to adenosine triphosphate (ATP), noradrenaline and acetylcholine were potentiated in a concentration dependent manner by dopamine in the presence of prazosin. This potentiation was not inhibited by raclopride, an antagonist for dopamine D2 and D3 receptors. However, the potentiation of ATP- and noradrenaline-induced contraction was inhibited by clozapine and 8-methyl-6-(4-methyl-1-piperazinyl)-11H-pyrido[2,3b][1,4]benzodiazepine (JL-18), dopamine D4 receptor antagonists. Further, the potentiation of noradrenaline- and acetylcholine-induced contraction was also inhibited by spiperone, an antagonist for dopamine D2, D3 and D4 receptors. These results suggest that the dopamine D4 receptor is located on the postsynaptic site of guinea pig vas deferens and that activation of the dopamine D4 receptor enhances contractile responses to agonists without affecting muscle tone.

Possible selective inhibition of [3H]adenosine uptake by papaverine in vascular adrenergic nerves.

The effect of the adenosine uptake inhibitors dipyridamole, papaverine and diazepam on the [3H]adenosine uptake was assessed using the rabbit pulmonary arterial segment. [3H]Adenosine uptake into the vascular segment was significantly diminished by dipyridamole (10(-6) -10(-5) M), papaverine (10(-5) -10(-4) M) or diazepam (5 x 10(-5) M) with potencies: dipyridamole greater than papaverine greater than diazepam. Dipyridamole (10(-6) M) or diazepam (5 x 10(-5) M) added 30 min before the incubation with [3H]adenosine reduced the high KCl-induced and epinephrine-induced [3H]purine effluxes equally, whereas papaverine (10(-5) M) selectively diminished the former efflux. KCl and epinephrine have been shown to act preferentially on the neuronal and extraneuronal sites, respectively. These results suggest that papaverine inhibits adenosine uptake into the vascular neuronal compartment, in preference to that into the extraneuronal compartment.

Existence of postsynaptic dopamine D2 receptor as an enhancer of contractile response in vas deferens.

Effects of dopamine and (+/-)-2-(N-phenylethyl-N-propyl)amino-5-hydroxy-tetralin hydrochloride (N-0434), a dopamine D2 receptor agonist, in the presence of prazosin on the ATP- and acetylcholine-induced contraction were investigated in the guinea-pig vas deferens in order to test for the existence of postsynaptic dopamine receptors. The contraction induced by ATP was potentiated by dopamine and N-0434. This potentiation was antagonized by spiperone, a dopamine D2 receptor antagonist, but not by a dopamine D1 receptor antagonist and an alpha2-adrenoceptor antagonist. Similar results were also observed by acetylcholine as well as ATP. The contraction induced by transmural nerve stimulation in the presence of alpha-adrenoceptor antagonists was also potentiated by N-0434, and this potentiation was antagonized by spiperone. The results suggest that dopamine D2 receptors are located on the postsynaptic site of guinea-pig vas deferens and that the contractile responses to ATP and acetylcholine are potentiated via activation of dopamine D2 receptor.

Clonidine-evoked selective P1-purinoceptor antagonism of contraction of guinea-pig urinary bladder.

The effect of clonidine on P1- and P2-purinoceptors of guinea-pig urinary bladder was compared to that of alpha, beta-methylene ATP, a selective P2-purinoceptor desensitizer. After, alpha, beta-methylene ATP, 10 microM, vesical contraction produced by ATP was eliminated while that caused by acetylcholine was unaffected. Clonidine, however, failed to antagonize ATP-induced contraction of the segment even at 100 microM. Electrically evoked contraction of the bladder was partly attenuated by 0.3 microM atropine and the remainder was markedly reduced by 3-30 microM alpha, beta-methylene ATP, suggesting an important role of ATP as an excitatory transmitter in this tissue. This stimulus-evoked contraction was also suppressed by adenosine, a P1-purinoceptor agonist, in a concentration-dependent fashion, and the suppression was greatly antagonized by 50 microM clonidine. These results suggest that the antagonistic property of clonidine is substantially selective for presynaptic P1-purinoceptors in contrast with that of alpha, beta-methylene ATP for postsynaptic P2-purinoceptors.

Opposite modulation by muscarinic M1 and M3 receptors of acetylcholine release from guinea pig ileum as measured directly.

Muscarinic receptor agonist and antagonist effects on acetylcholine release evoked by electrical or dimethylphenylpiperazinium stimulation from guinea pig ileum were evaluated by measuring acetylcholine with a high performance liquid chromatography-electrochemical detector system. AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine), a muscarinic M1 receptor agonist, increased markedly the evoked release of acetylcholine. In contrast, pirenzepine decreased the evoked acetylcholine release. 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine methiodide) and p-F-HHSiD (p-fluoro-hexahydrosiladifenidol), muscarinic M3 antagonists, increased the release of acetylcholine. Atropine enhanced acetylcholine release to a similar extent while bethanechol reduced the electrically evoked acetylcholine release. This reduction was virtually unaffected by methoctramine, but was antagonized by 4-DAMP or p-F-HHSiD. The results from direct determination of acetylcholine suggest that, in contrast to autoinhibition by stimulation of muscarinic M3 receptors, stimulation of presynaptic muscarinic M1 receptors is predominantly involved in enhancement of the acetylcholine release from guinea pig ileal nerves, and that AF102B functions as a muscarinic M1 agonist in this peripheral neuron.

Different mechanisms of the rabbit pulmonary arterial contraction caused by 5- or 6-hydroxydopamine.

The contractile responses of rabbit arterial strips to 5-hydroxydopamine (5-OHDA) or 6-hydroxydopamine (6-OHDA) were assessed in vitro. The 3H efflux from the pulmonary artery after preloading with [3H]noradrenaline was markedly enhanced by 100 microM 5-OHDA or 6-OHDA. In non-superfused strips, 6-OHDA produced a biphasic contraction, an initial small transient and a subsequent large sustained contraction, whereas 5-OHDA elicited a large monophasic contraction. The 6-OHDA-evoked second large contraction was followed by a marked tachyphylaxis after repeated application of the drug. This contraction was greatly diminished by prazosin or cocaine and by pretreatment with reserpine, indicating an indirect action via noradrenaline release. In contrast, the initial contraction caused by 6-OHDA as well as the contraction caused by 5-OHDA was not inhibited by these agents, except prazosin, implying a direct action at the postsynaptic alpha 1-adrenoceptors. In addition, the concentration-response curve for noradrenaline was significantly shifted to the right by pre-addition of 5-OHDA whereas the curve for 5-HT was virtually unaffected. The results thus suggest that 5-OHDA acts as a postsynaptic alpha 1-adrenoceptor agonist.