Exercise and the multidisciplinary holistic approach to adolescent dysautonomia.

AIM: To determine whether an eight-week strength training programme as part of a multidisciplinary approach would minimise symptoms and improve quality of life in patients with dysautonomia. METHODS: Adolescents referred to a tertiary-level cardiology service from May 2014-December 2015 with symptoms of dysautonomia were eligible. Participants completed an exercise test and a quality of life (QoL) questionnaire (PedsQL) prior to the intervention. Participants were asked to complete exercises five times per week. After eight weeks, participants returned for follow-up testing. Parents completed a proxy report of their child's QoL at both time points. RESULTS: A total of 17 participants completed the study protocol with an adherence rate of up to 50%. Post-intervention, QoL scores improved across all levels in the participants [total 65.2 (50.4-74.7) vs 48.9 (37.5-63.0); p = 0.006; psychosocial 65.8 (56.1-74.6) vs 50.0 (41.7-65.8); p = 0.010; physical 62.5 (37.5-76.6) vs 43.8 (25-68.5); p = 0.007] and their parent proxy reports [total 63.5 (48.7-81.3) vs 50.0 (39.3-63.0); p = 0.004; psychosocial 62.1 (52.1-81.3) vs 50.0 (39.6-59.2); p = 0.001; physical 62.5 (51.6-80.0) vs 50.0 (27.5-70.3); p = 0.003]. Treadmill time also improved (9.1 vs 8.0 minutes; p = 0.005). CONCLUSION: Following an eight-week strength training programme, dysautonomia patients report a significant improvement in both their quality of life and endurance time.

Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation.

The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated. Superfusate collected during EFS rapidly metabolised exogenous ATP (100 microM) and 50% was broken down in 5.67+/-0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 40 microM and V(max) of 20.3 nmol ATP metabolized min(-1) ml(-1) superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80 degrees C for 10 min. ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC(50)=25 microM (95% confidence limits=22-27 microM), Hill slope=-1.06+/-0.04). When EFS was applied three times at 30 min intervals, ATP metabolism was 20-30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz. In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent.

Electrophysiology of autonomic neuromuscular transmission involving ATP.

Electrophysiological investigations of autonomic neuromuscular transmission have provided great insights into the role of ATP as a neurotransmitter. Burnstock and Holman made the first recordings of excitatory junction potentials (e.j.p.s) produced by sympathetic nerves innervating the smooth muscle of the guinea-pig vas deferens. This led to the identification of ATP as the mediator of e.j.p.s in this tissue, where ATP acts as a cotransmitter with noradrenaline. The e.j.p.s are mediated solely by ATP acting on P2X(1) receptors leading to action potentials and a rapid phasic contraction, whilst noradrenaline mediates a slower, tonic contraction which is not dependent on membrane depolarisation. Subsequent electrophysiological studies of the autonomic innervation of smooth muscles of the urogenital, gastrointestinal and cardiovascular systems have revealed a similar pattern of response, where ATP mediates a fast electrical and mechanical response, whilst another transmitter such as noradrenaline, acetylcholine, nitric oxide or a peptide mediates a slower response. The modulation of junction potentials by a variety of pre-junctional receptors and the mechanism of inactivation of ATP as a neurotransmitter will also be described.

Characterization of the ATPase released during sympathetic nerve stimulation of the guinea-pig isolated vas deferens.

The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5'-triphosphate (ATP) acts as a neurotransmitter. Superfusate collected during EFS of sympathetic nerves of the guinea-pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea-pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea-pig isolated vas deferens. However, EFS of the enteric nerves of the guinea-pig isolated taenia coli did not release any detectable ATPase. The ATPase released from the guinea-pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37 degrees C and 20 degrees C. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 39 microM and a V(max) of 1039 pmol ATP metabolized min(-1) ml(-1) superfusate. 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and pyridoxal-5'-phosphate (P-5-P) all inhibited the ATPase activity in a concentration-dependent manner with a potency order of ARL 67156 = PPADS>P-5-P. In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea-pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases.

The effect of P2 receptor antagonists and ATPase inhibition on sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens.

1. Intracellular microelectrodes were used to record the transmembrane potential and excitatory junction potentials (e.j.p.s) produced by sympathetic nerve stimulation (1 Hz) in smooth muscle cells of the guinea-pig isolated vas deferens. 2. The symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid (NF023) produced a concentration-dependent inhibition of e.j.p. magnitude (IC(50)=4. 8x10(-6) M), but had no effect on the resting membrane potential of the smooth muscle cells. 3. Pyridoxal-5-phosphate (P-5-P) also depressed e.j.p. magnitude in a concentration-dependent manner, but was less potent than NF023 (IC(50)=2.2x10(-5) M). At 10(-4) M and above P-5-P significantly depolarized the smooth muscle cells. 4. The nucleoside triphosphatase inhibitor 6-N,N-diethyl-D-beta, gamma-dibromomethyleneATP (ARL 67156) (5x10(-5) M) significantly increased e.j.p. amplitude. ARL 67156 (10(-4) M) further increased e. j.p. amplitude such that they often reached threshold for initiation of action potentials, causing muscle contraction and expulsion of the recording electrode. 5. After reduction of e.j.p.s by NF023 or P-5-P (both 10(-5) M), subsequent co-addition of ARL 67156 (10(-4) M) significantly increased their magnitude. 6. The overflow of endogenous ATP evoked by field stimulation of sympathetic nerves (8 Hz, 1 min) was measured by HPLC and flurometric detection. ARL 67156 (10(-4) M) enhanced ATP overflow by almost 700% compared to control. 7. We conclude that for electrophysiological studies NF023 is preferable to other P2X receptor antagonists such as pyridoxalphosphate -6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or P-5-P. Furthermore, breakdown of endogenous ATP by nucleoside triphosphatases is an important modulator of purinergic neurotransmission in the guinea-pig vas deferens.

Modulation of purinergic neurotransmission.

During the past 25 years ATP has become accepted as an important neurotransmitter at a wide variety of neuroeffector junctions, usually acting as a cotransmitter with NA, ACh, nitric oxide or a neuropeptide such as NPY or VIP. The details of the storage and release of ATP with its cotransmitters has yet to be resolved. However, recent studies indicate that there is more than one population of storage vesicles in the nerves, since the release of the various cotransmitters varies over time and can be differentially modulated by drugs. The subclassification of P2 receptors has advanced dramatically in the past few years due to the use of molecular biology methods allowing the cloning and expression of 14 different subclasses of P2 receptors, seven P2X and seven P2Y. Determination of the functional significance of the various receptor subtypes would be helped by the development of selective agonists and antagonists. The neurotransmitter action of ATP at visceral and vascular smooth muscle P2X receptors has been elucidated in considerable detail. ATP induces a transient inward current via ligand-gated channels, which produces EJPs, action potentials and a phasic contraction of the effector tissue. ATP's neurotransmitter actions appear to be curtailed by the action of ATPases. It has been assumed that this ATPase activity is due to membrane bound ecto-ATPases on the surface of the effector tissue, however, the recently identified soluble ATPase released during nerve stimulation could also be involved in inactivation of ATP. The relative importance of ecto-ATPase and the releasable ATPase is yet to be determined.

Comparison of the actions of ATP and UTP and P(2X1) receptors in smooth muscle of the rat tail artery.

The actions of ATP and uridine 5'-triphosphate (UTP) were compared at P2X1 receptors in acutely dissociated smooth muscles cells of the rat tail artery. ATP (30 nM-100 microM) and UTP (1 microM-1 mM) elicited concentration-dependent inward currents. ATP was approximately 100-fold more potent than UTP. In both cases, currents were activated within 3 ms of agonist application and had similar time-courses of activation and inactivation. The decay of responses for both agonists was concentration-dependent and in most cells could be fitted by two exponentials. The P2X receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 5 microM) inhibited responses to both ATP and UTP. An action of UTP at P2X1 receptors has not previously been reported. However, since the responses to ATP and UTP had similar time-courses and as PPADS and suramin inhibited both agonists, it is concluded that ATP and UTP are acting at the same site in these cells, the P2X1 receptor.

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery.

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156). 2. Suramin (1 microM(-1) mM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (0.3-300 microM) inhibited contractions evoked by equi-effective concentrations of alpha,beta-methyleneATP (alpha,beta-meATP) (5 microM), 2-methylthioATP (2-meSATP) (100 microM) and adenosine 5'-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to alpha,beta-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS. 3. Contractions evoked by uridine 5'-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 microM) and potentiated by PPADS (300 microM). 4. Desensitization of the P2X1-receptor by alpha,beta-meATP abolished contractions evoked by 2-meSATP (100 microM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15+/-3% and 68+/-4% of control. 5. Responses to alpha,beta-meATP (5 microM) and 2-meSATP (100 microM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24+/-6% and 61+/-13%, respectively, of their control response. 6. ARL 67156 (3-100 microM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to alpha,beta-meATP (5 microM), 2-meSATP (100 microM) and ATP (1 mM) in a concentration-dependent manner. 7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X1-receptors and G-protein-coupled P2Y-receptors.

The ecto-ATPase inhibitor ARL 67156 enhances parasympathetic neurotransmission in the guinea-pig urinary bladder.

The influence of enzymatic degradation on the neurotransmitter actions of ATP was studied using the ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156). Field stimulation of the parasympathetic nerves innervating guinea-pig urinary bladder muscle strips (1-8 Hz for 20 s) produced characteristic biphasic contractions, the peak magnitudes of which were significantly increased by 29-32% by ARL 67156 (100 microM). A similar degree of enhancement was seen in the presence of atropine (1 microM), consistent with ARL 67156 acting to enhance the action of neuronally released ATP. The effects of ARL 67156 reversed rapidly on washout of the drug. Contractions evoked by exogenous ATP (100 microM) were also potentiated by ARL 67156 (100 microM), but those to the stable analogue alpha,beta-methyleneATP (5 microM) were unaffected. ARL 67156 (100 microM) also enhanced contractions to exogenous acetylcholine (1 microM) and histamine (3 microM), but this potentiation was abolished by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (100 mciroM). It is concluded that when ATP acts as a neurotransmitter its postjunctional actions are attenuated by enzymatic degradation. ARL 67156 inhibits this breakdown.

Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation.

Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.

The interaction of diadenosine polyphosphates with P2x-receptors in the guinea-pig isolated vas deferens.

1. The site(s) at which diadenosine 5',5"'-P1,P4-tetraphosphate (AP4A) and diadenosine 5', 5"'-P1,P5-pentaphosphate (AP5A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P2-receptor antagonists and the ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156). 2. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (300 nM - 30 microM), suramin (3-100 microM) and pyridoxal-5'-phosphate (P-5-P) (3-1000 microM) inhibited contractions evoked by equi-effective concentrations of AP5A (3 microM), AP4A (30 microM) and alpha,beta-methyleneATP (alpha,beta-meATP) (1 microM), in a concentration-dependent manner and abolished them at the highest concentrations used. 3. PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP5A, AP4A and alpha,beta-meATP with similar IC50 values. No significant difference was found between IC50 values for suramin against alpha,beta-meATP and AP5A or alpha,beta-meATP and AP4A, but suramin was more than 2.5 times more potent against AP4A than AP5A. P-5-P showed the same pattern of antagonism. 4. Desensitization of the P2xi-receptor by alpha,beta-meATP abolished contractions evoked by AP5A (3 microM) and AP4A (30 microM), but had no effect on those elicited by noradrenaline (100 microM). 5. ARL 67156 (100 microM) reversibly potentiated contractions evoked by AP4A (30 microM) by 61%, but caused a small, significant decrease in the mean response to AP5A (3 microM). 6. It is concluded that AP4A and AP5A act at the P2xi-receptor, or a site similar to the P2xi-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP4A, but not AP5A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.

Enhancement of sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens by the novel ecto-ATPase inhibitor ARL 67156.

1. Field stimulation of the sympathetic nerves of the guinea-pig isolated vas deferens with trains of pulses of 20 s at 1-8 Hz produced characteristic biphasic contractions. The effect of the novel ecto-ATPase inhibitor, 6-N,N-diethyl-D-beta, gamma-dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5'-triphosphate (ATP) on its action as a neurotransmitter. 2. The peak magnitude of the response to nerve stimulation was significantly increased in a concentration-dependent manner by ARL 67156 (5-100 microM) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 microM). 3. ARL 67156 (100 microM) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout. 4. The neurogenic contraction in the presence of prazosin (0.1 microM) was purely purinergic, as it was abolished by the P2-purinoceptor antagonist, PPADS (100 microM). ARL 67156 (100 microM) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP. 5. Exogenous ATP and alpha, beta-methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 microM), whereas those to the stable analogue, alpha, beta-methylene ATP were not significantly affected. 6. Contractions to exogenous noradrenaline (10 microM) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 microM), but this potentiation was abolished by PPADS (100 microM). Therefore, this effect of the ecto-ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists. 7. It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.

ATP as a co-transmitter with noradrenaline in sympathetic nerves--function and fate.

ATP and noradrenaline are co-stored in synaptic vesicles in sympathetic nerves and when co-released act postjunctionally to evoke contraction of visceral and vascular smooth muscle. In the original purinergic nerve hypothesis it was proposed that ATP would then be sequentially broken down to ADP, AMP and adenosine. Although such breakdown can be measured, it is not clear how the time-scale of breakdown compares with the time-course of the postjunctional actions of ATP. We have investigated the role of ectoATPase in modulating purinergic neurotransmission in the guinea-pig vas deferens using ARL67156 (formerly FPL67516), a recently developed inhibitor of ectoATPase. ARL67156 (1-100 microM) potentiated neurogenic contractions in a concentration-dependent manner. Onset of potentiation was rapid and the effect reversed rapidly on washout of the drug. The effect was also frequency dependent, being greater at lower frequencies. The purinergic component of the neurogenic contraction was isolated using the alpha 1 antagonist prazosin (100 nM) and ARL67156 caused a similar potentiation. ARL67156 also potentiated contractions evoked by exogenous ATP (100 microM), but had no effect on those of the stable analogue alpha, beta-methylene ATP (500 nM). In the presence of the P2 purinoceptor antagonist PPADS (100 microM), ARL67156 also had no effect on contractions evoked by noradrenaline (10 microM) or KCI (40 mM). These results are consistent with an inhibitory action of ARL67156 on ectoATPase and suggest that ectoATPase modulates purinergic transmission in the guinea-pig vas deferens. When released from sympathetic nerves, ATP acts at the P2X purinoceptor, a ligand-gated cation channel, to evoke depolarization and contraction. In single acutely dissociated smooth muscle cells of the rat tail artery, studied under voltage-clamp conditions, ATP and its analogues evoke an inward current, with a rank order potency of 2-methylthioATP = ATP > alpha, beta-methylene ATP. This is very different from the order of potency for evoking contraction in whole vessel rings, which is alpha, beta-methylene ATP > > 2-methylthioATP > or = ATP. This discrepancy can be explained by a previously unrecognized attenuation of the action of ATP and 2-methylthioATP, but not alpha, beta-methylene ATP, by ectoATPase in whole tissues.

The effects of suramin on purinergic and noradrenergic neurotransmission in the rat isolated tail artery.

Intracellular microelectrode recording was used to examine the effects of suramin, a P2-purinoceptor antagonist, on the electrical responses evoked by sympathetic nerve stimulation in the rat isolated tail artery. Field stimulation (10 or 20 pulses at 0.5, 1 and 2 Hz) evoked a biphasic electrical response, consisting of fast, transient excitatory junctional potentials (e.j.p.s) and a slow, prolonged depolarisation. Suramin (100 microM) abolished the e.j.p.s and significantly increased the amplitude of the slow depolarisation at all frequencies. In contrast, phentolamine (2 microM) abolished the slow depolarisation, but had no effect on the magnitude of e.j.p.s. Neither drug altered the resting membrane potential of cells. The ability of suramin to inhibit e.j.p.s in rat tail artery is consistent with the proposal that it is a P2X-purinoceptor antagonist and supports a role for ATP as an excitatory cotransmitter from the sympathetic nerves innervating this tissue. Suramin is also able to increase the alpha-adrenoceptor-mediated slow depolarisation by an unknown mechanism.

Investigation of the actions of PPADS, a novel P2x-purinoceptor antagonist, in the guinea-pig isolated vas deferens.

1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) was investigated for its ability to act as an antagonist at P2x-purinoceptors which mediate neurogenic excitatory junction potentials (e.j.ps) and contractions in the guinea-pig isolated vas deferens. 2. PPADS (10(-7) M) caused a small potentiation of the phasic, predominantly purinergic component of contractions evoked by symapthetic nerve stimulation, but higher concentrations of PPADS (3 x 10(-6)-3 x 10(-5) M) elicited a substantial and significant concentration-dependent inhibition. In contrast, over the same concentration-range, PPADS had no effect on the tonic, predominantly noradrenergic phase. 3 PPADS (3 x 10(-5) M) also inhibited contractile responses to exogenous alpha,beta-methyleneATP (10(-8)-10(-3)M), a P2x-purinoceptor agonist, without affecting the responses to exogenous noradrenaline (10(-8)-10(-3) M), carbachol (10(-5) M) or histamine (10(-4) M). 4. PPADS (10(-7)-3 x 10(-5) M) produced a concentration-dependent reduction in e.j.p. magnitude and resting membrane potential. The maximum effect was seen at 10(-5) M PPADS, which reduced e.j.p. magnitude from 13.7 +/- 0.6 mV (n = 12) to 1.8 +/- 0.7 mV (n = 12) and membrane potential from -64.8 +/- 0.6 mV (n = 51) to -55.0 +/- 1.8 mV (n = 12). 5. The PPADS-induced depolarization was not inhibited by the P2x-purinoceptor antagonist, suramin (10(-4) M). This indicates that the depolarization was not due to an agonist action of PPADS at P2x-purinoceptors. 6. The results support the proposal that PPADS is a selective antagonist at P2x purinoceptors as opposed to non-P2-purinoceptors in the guinea-pig vas deferens, but its ability to cause membrane depolarization independently of P2x-purinoceptors and also, at a low concentration, to potentiate the phasic component of the neurogenic contraction indicates that it has other actions.

Evidence for nitric oxide as an inhibitory neurotransmitter in rabbit isolated anococcygeus.

The role of nitric oxide (NO) and ATP as putative inhibitory non-adrenergic, non-cholinergic (NANC) neurotransmitters was investigated in rabbit isolated anococcygeus after block of adrenergic and cholinergic responses, and raising tone with histamine. NANC nerve stimulation produced rapid relaxations which were completely abolished by tetrodotoxin. The magnitude of the NANC inhibitory responses was significantly reduced by the nitric oxide synthase inhibitors NG-nitro-L-arginine (NO-Arg) and NG-nitro-L-arginine methyl ester (L-NAME). This effect could be partially reversed by L-arginine but not by D-arginine. Oxyhaemoglobin inhibited NANC nerve responses and sodium nitroprusside mimicked the effects of NANC nerve stimulation. NO-Arg also reduced the magnitude of the inhibitory junction potentials recorded from the smooth muscle cells during NANC nerve stimulation. Exogenously applied ATP and adenosine each produced concentration dependent relaxations which were unaffected by the NO-synthase inhibitor NO-Arg. Relaxations to adenosine were virtually abolished by the P1 purinoceptor antagonist 8-(p-sulphophenyl)theophylline. Relaxations to ATP were also significantly reduced, indicating that part of the response to exogenous ATP is due to its breakdown to adenosine and subsequent action on P1 purinoceptors. Relaxations of the tissue to ATP and adenosine were unaffected by the P2 purinoceptor antagonist suramin. NANC nerve mediated responses were not significantly changed by either 8-(p-sulphophenyl)theophylline or suramin. These results suggest that NO is involved in inhibitory NANC neurotransmission in the rabbit isolated anococcygeus, but do not support a role for ATP as a NANC neurotransmitter in this tissue.

Endothelium-dependent vasodilator responses of the isolated mesenteric bed are preserved in long-term streptozotocin diabetic rats.

Endothelium-dependent and endothelium-independent responses to exogenous vasoactive substances were compared in isolated, perfused mesenteric beds from control rats and in rats subjected to prolonged (15-17 weeks) streptozotocin induced diabetes. The main aim of the study was to determine whether the prolonged period of diabetes altered vascular endothelial function, and thereby modified vascular responsiveness to vasoconstrictors or vasodilators. Noradrenaline induced vasoconstriction was not significantly altered in preparations from diabetic rats compared to control. Vasodilator responses to acetylcholine (ACh) and ATP were endothelium-dependent, since they were greatly reduced or abolished after endothelium removal by perfusion with 0.1% Triton-X 100. The vasodilator action of these agents was fully preserved in the diabetic animals. Sodium nitroprusside produced a vasodilation which was endothelium-independent, this vasodilation was also preserved in the diabetic animal. We conclude that prolonged streptozotocin-induced diabetes does not reduce the ability of the mesenteric vascular endothelium to release vasodilator substances, nor does it alter the responsiveness of the bed to exogenous endothelium-independent vasodilator sodium nitroprusside or vasoconstrictor noradrenaline.

Regional variation in purinergic and adrenergic responses in isolated vas deferens of rat, rabbit and guinea-pig.

1. Electrical and mechanical responses of epididymal and prostatic regions of rat, rabbit and guinea-pig vas deferens have been examined to investigate regional variation in purinergic and adrenergic mechanisms. 2. Noradrenaline was significantly more potent in producing contraction in epididymal segments of the muscle than in prostatic segments. 3. ATP and alpha,beta,methylene ATP were significantly more potent in producing contraction of prostatic segments than epididymal segments. 4. In guinea-pig vas deferens the resting membrane potential was greater in smooth muscle cells in the prostatic region than in the epididymal. Excitatory junction potentials (EJPs) in both the epididymal and prostatic regions were of similar magnitude and were almost abolished by the P2x-purinoceptor antagonist suramin. 5. The alpha-adrenoceptor antagonist phentolamine had no inhibitory action on EJPs in either region of the guinea-pig vas deferens.