Functional consequences of inhibition of nucleotide breakdown in rat vas deferens: a study with Evans blue

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [³H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methylthio ATP (all 100 M) over 30 min. Evans blue (100 M) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order , \-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 M) shifted the concentration-response curve of , \-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > , \-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 M) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an — albeit impure — ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.     

P2-purinoceptor antagonists discriminate three contraction-mediating receptors for ATP in rat vas deferens

The sites of action at which ATP elicits contraction of the rat vas deferens were studied by means of the P₂-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), suramin and reactive blue 2.Increasing concentrations of PPADS (up to 1 mM), suramin (up to 1 mM) and reactive blue 2 (up to 320 M) reduced and eventually abolished contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP 3 M with IC₅₀ values of 2.1, 10.1 and 27.0 M, respectively. In contrast, PPADS and suramin caused only a partial inhibition of contractions elicited by ATP 1 mM, maximal reduction by about 40%, IC₅₀ values 1.3 and 5.0 M, respectively; reactive blue 2 did not change ATP-induced contractions. In tissues exposed to PPADS 320 M throughout, increasing concentrations of reactive blue 2 or suramin decreased contractions elicited by ATP 1 MM, IC₅₀ values 2.6 and 14.5 M, respectively. In tissues exposed to suramin 320 M throughout, increasing concentrations of PPADS decreased contractions elicited by ATP 1 mM, IC₅₀ 37.9 M, whereas reactive blue 2 slightly enhanced these contractions. In tissues exposed to reactive blue 2 100 M throughout, increasing concentrations of PPADS reduced contractions elicited by ATP 1 MM, IC₅₀ 26.6 M, whereas suramin caused no change. Pre-exposure to ,-methylene ATP 1 M to desensitize P_2X-purinoceptors reduced the response to ATP 1 mM by 91% in otherwise untreated tissues, but did not reduce the response to ATP 1 mM in tissues exposed throughout to PPADS 320 M, suramin 320 M or reactive blue 2 100 M. Neither PPADS nor suramin nor reactive blue 2 altered contractions elicited by KCl 35 mM. The P₁-purinoceptor antagonist 8-(p-sulfophenyl) theophylline 100 M did not change contractions elicited by ,-methylene ATP 3 M or ATP 1 mM.It is concluded that ATP 1 mM elicits contraction of the rat vas deferens through three sites: P_2X-purinoceptors which are blocked by PPADS, suramin and reactive blue 2; P_2Y-purinoceptors blocked by reactive blue 2 and suramin but resistant to PPADS; and non-P_2X-non-P_2Y-purinoceptors blocked by PPADS but resistant to inhibition by suramin and reactive blue 2. Correspondence to: R. Bültmann at the above address     

Two relaxation-mediating P2-purinoceptors in guinea-pig taenia caeci

2-Methylthio ATP, noradrenaline, adenosine 5-O-(2-thiodiphosphate) (ADP\S), ,\-methylene ATP, ATP and adenosine caused relaxation of guinea-pig isolated taenia caeci precontracted by carbachol, with potency decreasing in the order indicated. 4,4-Diisothiocyanatostilbene-2,2-disulphonate (DIDS) 10, 32 and 100 M shifted the concentration-response curve of ,\-methylene ATP increasingly to the right, by the factor 141 at DIDS 100 M. Concentration-response curves of the other agonists were not shifted to the right by DIDS 100 M, except for the curve of ADP\S which was shifted by the factor 2.4. The relaxation produced by 2-methylthio ATP faded rapidly. When the fade was complete, further addition of 2-methylthio ATP or ATP did not elicit relaxation, whereas the relaxant effect of , \-methylene ATP was unchanged. The results indicate that there are at least two relaxation-mediating P₂-purinoceptors in guinea-pig taenia caeci, the P_2Y-purinoceptor which is relatively insensitive to DIDS and a distinct receptor for ,\-methylene ATP which is very sensitive to DIDS.     

Inhibition by nucleotides acting at presynaptic P2-receptors of sympathetic neuro-effector transmission in the mouse isolated vas deferens

Summary Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [³H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S₁, S₂)., \-Methylene-ATP, ATPS, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mol/1) and uridine (up to 1 mmol/1). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATPS > ATP = adenosine > UTP; ,\-meth-ylene-ATP (up to 10 µmol/l) and uridine (up to 1 mmol/1) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATPS were not significantly attenuated. After prolonged exposure to ,-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2.It is concluded that ATP, ATPS, ,\-methylene-ATP and UTP produce contraction of the vas deferens by activation of P_2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P₂-receptor which differs from the P_2X-receptor and may be a reactive blue 2-resistant P_2y-like receptor.     

Corelease of noradrenaline and ATP by brief pulse trains in guinea-pig vas deferens

Contractions and overflow of tritium and ATP elicited by single electrical pulses or short pulse trains were studied in the guinea-pig isolated vas deferens preincubated with [³H]-noradrenaline. ATP was measured using the luciferase technique.A single pulse caused only a small contraction and minimal tritium and ATP overflow. In contrast, trains of 6 pulses elicited marked contractions as well as tritium and ATP overflow. In experiments with 6 pulses/100 Hz, prazosin 0.3 M reduced the contraction by 73 %, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 85%. Suramin 300 M reduced the contraction by 69% but changed neither the evoked overflow of tritium nor that of ATP. The combination of prazosin 0.3 gM and suramin 300 M abolished the contraction, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 70%. When 6 pulses were applied at frequencies of 1, 2, 10 or 100 Hz, all responses increased with frequency up to a maximum at 10 Hz, but contractions and the evoked overflow of ATP increased with frequency to a greater extent than the evoked overflow of tritium. A similar frequency overflow relationship was observed when the medium contained prazosin 0.3 M and suramin 300 M (and evoked ATP overflow was greatly reduced). Yohimbine 1 M did not affect the overflow of tritium evoked by 6 pulses/100 Hz but increased that evoked by 6 pulses/10 Hz.The results demonstrate an overflow of both noradrenaline and ATP in response to short pulse trains. As observed previously for prolonged pulse trains, the major part of the evoked overflow of ATP was derived from non-neural cells. The ATP overflow remaining during ₁-adrenoceptor blockade by prazosin and P₂-purinoceptor blockade by suramin is likely to reflect neural release of ATP. The results support the view that release of ATP increases with frequency to a greater extent than release of noradrenaline. The latency for the onset of prejunctional ₂-autoinhibition in guinea-pig vas deferens is between 50 and 500 ms. Correspondence to: I. von Kügelgen at the above address     

Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ‘fear’-motivated behaviour

Summary An elevated X-maze with alternating open and enclosed arms was investigated as a model for the study of fear-induced behaviour. As predicted, the anxiolytics diazepam and amylobarbitone increased, and the putative anxiogenics ACTH and picrotoxin decreased the proportion of open arm entries. The ₁-adrenoceptor agonists phenylephrine and ST587, and the ₂-adrenoceptor antagonists idazoxan, piperoxane, RS-21361 and yohimbine decreased relative open-arm entries, thus resembling the putative anxiogenics. On the other hand, azepexole, clonidine and guanabenz, agonists at ₂-adrenoceptors, and the ₁-adrenoceptor antagonists prazosin and thymoxamine, enhanced the proportion of open arm entries at low doses, suggesting anxiolytic-like properties. A paradoxical fall in open arm entries occurred with these agents at higher doses. These results provide further evidence for the involvement of noradrenergic systems in fear-motivated behaviour.     

Interaction of adenine nucleotides,UTP and suramin in mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect of ATP

Summary Effects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. ,-Methylene-ATP, ATPS, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 mol/l). Prolonged incubation with ,-methylene-ATP (concentration increased stepwise from 0 to 15 mol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10–300 mol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for ,-methylene-ATP, ATPS, UTP and lower concentrations of ATP (0.1–1 ol/l) to the right. The pA₂-values of suramin were 5.2 against ,-methylene-ATP, 4.8 against ATPyS, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P_2X-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P₂-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P_2X-receptor.Send offprint requests to I. von Kügelgen at the above address     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

Electrically-evoked release of taurine in the rat vas deferens: evidence for a purinoceptor-mediated effect

Release of taurine evoked by electrical stimulation (2700 pulses; 5 Hz; 10 mA unless stated otherwise) and its dependence on noradrenaline and ATP was studied in isolated, perifused rat vas deferens. Outflow of noradrenaline was also measured in some experiments.The basal outflow of taurine averaged 3.90±0.32 nmol/g tissue per min. Electrical stimulation increased the outflow to about 4 times basal values. The electrically-evoked overflow averaged 128.0±11.7 nmol/ g. An increase in current strength to 40 mA increased the evoked overflow by about 50%. At either current strength, the evoked overflow of taurine (and noradrenaline) was abolished by tetrodotoxin. Ca²⁺-deprivation blocked the overflow of taurine elicited by 10 mA and increased the overflow elicited by 40 mA pulses (but abolished noradrenaline overflow under either condition). Neither prazosin nor pretreatment of the rats with reserpine reduced electrically-evoked overflow of taurine (although reserpine pretreatment abolished evoked noradrenaline overflow). Tyramine (100 mol/1; 9 min) caused an overflow of taurine 36% of that caused by electrical stimulation (but an overflow of noradrenaline 3 times higher than that evoked by electrical stimulation). Exogenous noradrenaline (9 min) caused a concentration-dependent overflow of taurine with a maximal effect at 162 mol/1, amounting to 33% of the electrically-evoked overflow. ,\-Methylene ATP (19 mol/1) elicited an overflow of taurine that faded despite continued exposure to the drug and amounted to 62% of the response to electrical stimulation. Thirty minutes after the start of application of , \.-methylene ATP, electrically-evoked overflow of taurine was greatly reduced. Suramin (100 mol/1) also reduced taurine overflow in response to electrical stimulation.It is concluded that electrical (neural) stimulation releases taurine in rat vas deferens. The release is mainly postjunctional in origin, secondary to ATP release from sympathetic axon terminals, and a consequence of postjunctional P_2x-purinoceptor activation.     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions,G protein and adenylate cyclase

The effects of ₂-adrenoceptor agonists (dexmedetomidine, oxymetazoline), alone or in combination with various -adrenoceptor subtype-selective antagonists (CH-38083, idazoxan, WB4101, BRL44408, ARC-239, prazosin), on noradrenaline- and isoprenaline-induced lipolysis were investigated in human isolated abdominal subcutaneous fat cells. The rank order of potency of antagonists in preventing dexmedetomidine- and oxymetazoline-evoked suppression of isoprenaline-induced lipolysis was (pA₂-values): CH-38083 (7.69 and 7.48) idazoxan (7.5 and 7.41) > BRL 44408 (7.23 and 7.19) WB 4101 (7.13 and 7.12) > prazosin (5.18 and 5.17) > ARC-239 (4.72, 4.9). While CH-38083 and idazoxan, non-subtype selective ₂-adrenoceptor antagonists and BRL44408, a selective a_2A-adrenoceptor antagonist as well as WB4101 potentiated the lipolytic effect of noradrenaline, ARC-239, the selective _2B-adrenoceptor antagonist failed to affect it. In addition since the _2A-adrenoceptor selective agonist, oxymetazoline concentration dependently inhibited the lipolytic effect of isoprenaline, and WB4101 and BRL44408 (a_2A-adrenoceptor antagonists) antagonised the effect of oxymetazoline in a competitive manner, it is concluded that the a_2A-adrenoceptor subtype is involved in antilipolysis. In addition, functional evidence was obtained that there is an interaction between _2A- and -adrenoceptors located on the cell surface of adipocytes, through which locally released noradrenaline and/or circulating circulating adrenaline influence lipolysis.On leave from Institute of Medical Pharmacology, University of Pisa, Via Roma 55, I-5626 Pisa, Italy Correspondence to: E. S. Vizi at the above address     

Inhibition of presynaptic alpha-2-adrenoceptor and opioid receptor agonist responses in the rat vas deferens by chronic imipramine treatment

Summary The effects of chronic imipramine administration on agonist responses in rat isolated smooth muscle preparations were investigated. The administration of 20 mg/kg imipramine two times a day for 4 and 11 days resulted in an equivalent subsensitivity (approximately 8-fold) of clonidine-induced inhibition of electrically evoked contractions in the rat vas deferens (presynaptic ₂-adrenoceptor response). Imipramine (4 days) resulted in a marked inhibition of the ability of [d-Ala², d-Leu⁵] enkephalin to decrease electrically evoked contractions of the vas deferens (presynaptic opioid receptor response) but did not significantly affect the carbachol-induced increase in electrically evoked contractions (muscarinic receptor response). In the absence of cocaine the contractile effects of norepinephrine and tyramine in the vas deferens were, respectively, potentiated and inhibited, following imipramine (4 days), suggesting a decrease in the activity of the neuronal uptake mechanism. When determined in the presence of cocaine, the potency of the postsynaptic effects of norepinephrine in the vas deferens (₁-adrenoceptor response) was not significantly altered by imipramine (4 days). With regard to other postsynaptic receptors, imipramine (4 days) decreased slightly the potency of phenylephrine in the aorta (₁-adrenoceptor response) and increased slightly the potency of carbachol in the trachea (muscarinic receptor response) and the potency of serotonin in the rat aorta (5HT₂-receptor response). Thus, chronic imipramine administration decreased the potency of presynaptic ₂- and opioid agonist responses in the vas deferens but caused very little or no changes in the potencies of agonists at postsynaptic sites. Send offprint requests to R. A. Keith at the above address     

Reverse Iontophoresis: Noninvasive Glucose Monitoring in Vivo in Humans

Purpose. To demonstrate that reverse iontophoresis can be used to noninvasively obtain information about systemic glucose levels in vivo in humans. Methods. The passage of current across the skin in vivo drives ions into the tissue, from the electrode chambers positioned on the skin surface, and simultaneously pulls ions from the body in the opposite direction. Because of the net negative charge on the skin, under normal conditions, the membrane is permselective to cations, and a potential gradient also results, therefore, in electroosmotic convection of solvent in the direction of counterion flow (i.e., from anode to cathode). Thus, it is also possible to enhance the transport of polar, yet uncharged, species using iontophoresis. In an earlier study, the in vitro extraction of glucose, by reverse iontophoresis was established, and extension of the approach to an in vivo model was indicated. The idea has therefore been further explored in vivo in humans. Results. Using small, simple, prototypical electrode chambers, attached to the ventral forearm surface, direct current iontophoresis at 0.25 mA/cm² for periods of up to 1 hour, and a sensitive analytical procedure to measure the quantities of glucose extracted, it has been shown that iontophoretic sampling of glucose is feasible. However, the shorter periods (15 minutes or less) of extraction considered yield results which are contaminated (it is believed) by glucose that is a product of lipid metabolism within the skin. While this material is expected to complicate the initial calibration of the approach, the problem is effectively resolved within one hour, by which time the glucose arriving in the electrode chambers on the skin surface is expected to directly reflect the subcutaneous tissue concentration. Conclusions. Based upon these initial observations, further investigation can now be directed towards optimization of electroosmotic flow and sampling time, improved reproducibility and the development of a practical assay methodology.     

Effects of pertussis toxin on opioid regulation of catecholamine release from rat and guinea pig brain slices

Summary Opioid agonists selective for µ-, -, and -receptors are all capable of regulating the stimulated release of noradrenaline from three terminal fields (cortex, hippocampus, and cerebellum) of the noradrenergic projections from locus coeruleus in the guinea pig brain. Intracerebroventricular injections of pertussis toxin abolished the ability of a µ-selective agonist and of a -selective agonist to inhibit stimulated noradrenaline release, but left unaffected the concentration-related inhibition of NE release by a agonist. Thus, µ- and -receptors have been shown to be coupled to their effector systems in these noradrenergic neurons via guanyl nucleotide binding proteins (G proteins) which are sensitive to pertussis toxin, while -receptors in the same neurons appear to be coupled through a different mechanism which is significantly less sensitive to pertussis toxin. In contrast to opioid receptor regulation of noradrenaline release in guinea pig hippocampus, µ-, but not - or -agonists are capable of regulation of stimulated noradrenaline release from rat hippocampus and cortex, and -, but not µ- or -agonists are capable of inhibiting the stimulated release of dopamine from rat striatum and cortex. Pertussis toxin injections significantly attenuated µ-agonist inhibition of noradrenaline release, but had no effect on the ability of a -selective agonist to regulated dopamine release, confirming the insensitivity of the -receptor-effector coupling system to pertussis toxin.This work was supported by a grant from the National Institute on Drug Abuse. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. Animals used in this study were acquired and cared for in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 85-23 Revised 1985) Send offprint requests to L. L. Werling at the above address     

Selective enhancement by an adenosine A1 receptor agonist of agents inducing contraction of the rat vas deferens

The adenosine analogue N⁶-cyclopentyladenosine (CPA), acting via postjunctional A₁ receptors, has been shown to enhance contractions of the rat vas deferens induced by adenosine 5-triphosphate (ATP), the sympathetic cotransmitter in this tissue. The aim of the present study was to examine the ability of CPA to enhance contractions induced by other contractile agents. CPA (0.01–0.3 M) enhanced contractions induced by exogenous ATP (10 M), 5-hydroxytryptamine (5-HT) (3 M), tyramine (10 M), 2-methyl-5-hydroxytryptamine (2-Me-5-HT) (10 M) and KCl (35 mM) and this enhancement was blocked by an A₁-selective concentration (3 nM) of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CPA failed to enhance contractions induced by exogenous noradrenaline (NA) (1 M or 10 M), bradykinin (0.1 M), phenylephrine (3 M) or carbachol (10 M).The contractions induced by ATP (10 M), 5-HT (3 M), 2-Me-5-HT (10 M) and KCl (35 mM) were unaffected by tetrodotoxin (1 M) as well as by desensitisation of the P_2x-purinoceptors with the ATP analogue adenosine 5-(, -methylene) triphosphonate. The contractions induced by tyramine (10 M) and 2-Me-5-HT (10 M) were blocked by prazosin (100 nM) or by imipramine (1 M). Ketanserin (10 nM) antagonised the response to 5-HT giving a dose-ratio of 12.9 corresponding to an apparent pA₂ of 9.1.In conclusion, the A₁-mediated effect was clearly selective for certain contractile agents and not due to a non-specific increase in contractility of the tissue. CPA enhanced contractions induced by both ATP and indirect sympathomimetics which release endogenous NA, and this enhancement of the two sympathetic cotransmitters may have a functional significance, and demonstrates the complexity of the neuromodulatory effects of adenosine in the rat vas deferens.     

Inhibition of Nuclear Transcription Factor-κB by Specific IκB Kinase Peptide Inhibitor

Pharmaceutical Research -     

Responses to catecholamines of the rat isolated uterus throughout the natural oestrous cycle

Summary The effects of noradrenaline (NA), adrenaline (Adr) and isoprenaline (Iso) on rat isolated uterus were studied, throughout the natural oestrous cycle. The -inhibitory effects of the catecholamines were measured as a percentage inhibition of a standard acetylcholine (ACh)-induced contraction. Iso produced approximately 80% maximum inhibition of the standard ACh-contraction in all 4 stages of the oestrous cycle. Adr and NA produced 80% maximum inhibition in oestrus only, and 50–60% maximum inhibition in proestrus, metoestrus and dioestrus. The differences in the degree of inhibition produced by the catecholamines were overcome (i.e. the maximum inhibition produced by Adr and NA was increased to become equal to that produced by Iso) when experiments were repeated in the presence of the uptake inhibitors desmethylimipramine (DMI) and normetanephrine (NMN), but not in the presence of an -adrenoceptor antagonist (azapetine 10^–7 M). Variations in the uptake of³H-Iso and³H-NA were observed in the different stages of the oestrous cycle. Small -adrenoceptor mediated motor responses to NA and Adr were observed only in the presence of a -antagonist (propranolol 10^–5 M), in uteri from rats in oestrus, metoestrus and dioestrus, but not proestrus. It is concluded that in the rat isolated uterus, -inhibitory receptors predominate throughout the natural oestrous cycle, although the existence of -excitatory receptors has been shown in 3 of the 4 stages.     

In vivo studies on alpha-adrenergic receptor subtypes in human veins

Summary We studied in vivo responsiveness of venous ₁ and ₂-adrenoceptors, measuring the diameter changes in superficial veins in response to -adrenergic agonists and antagonists in healthy human volunteers. The dorsal hand vein technique was used because it permits complete dose-response studies of venous constriction without confounding reflex alterations.Local infusions of all agonists studied induced dose-dependent contraction of the hand vein; the maximal effects (Emax) were: norepinephrine (88% ± 10%), methox amine (97% ± 5%), phenylephrine (95% ± 6%), clonidine (54% ± 12%), and azepexole (68% ± 26%). Clonidine reduced the norepinephrine-induced venoconstriction by 11% ± 10%. Oral doses of 1 mg prazosin antagonized the venoconstriction induced by norepinephrine, methoxamine, and clonidine, but not by azepexole. Yohimbineantagonism was observed against all agonists studied. Inhibition by yohimbine of clonidine-induced venoconstriction was irreversible over 60–180 min.Results show that the in vivo effects on veins of -adrenergic agonists are in good agreement with results from in vitro experiments. Agonists with ₁- and ₂-adrenoceptor subtype selectivity cause venoconstriction in vivo, but ₂-receptor mediated constriction is intrinsically weaker. Clonidine acts as a partial antagonist against norepinephrine, presumably on postsynaptic ₂-receptors. At high doses, ₂-adrenoceptor subtype selectivity of clonidine and yohimbine appear to be partially lost in vivo. Send offprint requests to H. G. Eichler at the above address     

Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens

Summary Effects of electrical stimulation and nicotine on ATP and tritium outflow and smooth muscle tension were studied in the guinea-pig isolated vas deferens preincubated with [³H]-noradrenaline. ATP was measured using the luciferase technique.Electrical stimulation caused biphasic contractions and an acceleration of ATP and tritium outflow. The contraction amplitude and the overflow of ATP increased markedly, whereas the overflow of tritium increased only slightly with the frequency of stimulation (1–10 Hz; constant number of 60 pulses). The contraction amplitude did not increase with an increase in pulse number (20–540 pulses; constant frequency of 5 Hz), whereas the overflow of ATP increased slightly, and that of tritium markedly. Nicotine caused monophasic, transient contractions and, again, an acceleration of ATP and tritium outflow. Contractions, ATP and tritium overflow increased with the concentration of nicotine (56–320 mol/l) in an approximately parallel manner. The influence of some drugs on responses to electrical stimulation (60 pulses, 5 Hz) and nicotine (180 mol/l) was investigated. Tetrodotoxin blocked all effects of electrical stimulation but did not change those of nicotine. The reverse was true for hexamethonium. Neither electrical stimulation nor nicotine caused contraction or an increase in ATP outflow after pretreatment with 6-hydroxydopamine. The main effects of prazosin 0.3 mol/l were to reduce electrically evoked contractions (above all second phase) as well as nicotine-evoked contractions and the nicotine-evoked overflow of ATP (the latter by about 81 %). Prazosin also tended to diminish the electrically evoked overflow of ATP. ,ß-Methylene-ATP 10 mol/l elicited a transient contraction and ATP overflow on its own. The main change in the subsequent state of desensitization was a decrease of the first phase of electrically evoked contractions. The main effects of prazosin combined with desensitization by ,ß-methylene-ATP were marked decreases of electrically evoked contractions (by 94%), the electrically evoked overflow ATP (by 66%), nicotine-evoked contractions (by 97%) and the nicotinee-voked overflow of ATP (by 70%).It is concluded that both electrical stimulation and nicotine release noradrenaline and ATP in guinea-pig vas deferens. Only part of the evoked overflow of ATP (about 32%) is neural in origin. Another part probably originates from smooth muscle cells where it is released by neurogenic noradrenaline acting at ₁-adrenoceptors. Corelease leads to cotransmission: electrically as well as nicotine-evoked contractions consist of adrenergic and purinergic components. Varying types of stimulation release cotransmitter mixtures of varying composition. Electrical stimulation at high frequency (for example 10 Hz) and with low pulse numbers (for example 20 pulses) seems to release the cotransmitters at a relatively high ATP/noradrenaline ratio. Activation of prejunctional nicotine receptors seems to release the cotransmitters at a relatively low ATP/noradrenaline ratio. Send offprint requests to Ivar von Kügelgen at the above address     

On the role of α-methyldopamine in the antihypertensive effect of α-methyldopa

Summary In renal hypertensive rats the cerebral concentration of -methyldopa, -methyldopamine, -methylnoradrenaline, dopamine and noradrenaline as well as the blood-pressure were determined simultaneously. The antihypertensive effect followed a time course identical to that of the increase in the cerebral concentration of -methyldopamine and of the decrease in the concentration of dopamine, whereas lowering of blood pressure on the one hand, and changes in the levels of -methylnoradrenaline and noradrenaline, on the other, were not related to each other. Dose-response relationships showed the same correlations and lack of correlations, respectively.These results suggest that non--hydroxylated catecholamines play a major role in mediating the antihypertensive effect of -methyldopa or, alternatively, that only the newly biosynthesized -methyl-noradrenaline is effective in lowering blood pressure.A preliminary communication has been presented at the Spring Meeting 1973 of the German Pharmacological Society at Mainz (Waldmeier et al., 1973).