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There is nervous control of the heart by ATP as a cotransmitter in sympathetic, parasympathetic, and sensory-motor nerves, as well as in intracardiac neurons. Centers in the brain control heart activities and vagal cardiovascular reflexes involve purines. Adenine nucleotides and nucleosides act on purinoceptors on cardiomyocytes, AV and SA nodes, cardiac fibroblasts, and coronary blood vessels. Vascular tone is controlled by a dual mechanism. ATP, released from perivascular sympathetic nerves, causes vasoconstriction largely via P2X1 receptors. Endothelial cells release ATP in response to changes in blood flow (via shear stress) or hypoxia, to act on P2 receptors on endothelial cells to produce nitric oxide, endothelium-derived hyperpolarizing factor, or prostaglandins to cause vasodilation. ATP is also released from sensory-motor nerves during antidromic reflex activity, to produce relaxation of some blood vessels. Purinergic signaling is involved in the physiology of erythrocytes, platelets, and leukocytes. ATP is released from erythrocytes and platelets, and purinoceptors and ectonucleotidases are expressed by these cells. P1, P2Y 1 , P2Y 12 , and P2X1 receptors are expressed on platelets, which mediate platelet aggregation and shape change. Long-term (trophic) actions of purine and pyrimidine nucleosides and nucleotides promote migration and proliferation of vascular smooth muscle and endothelial cells via P1 and P2Y receptors during angiogenesis, vessel remodeling during restenosis after angioplasty and atherosclerosis. The involvement of purinergic signaling in cardiovascular pathophysiology and its therapeutic potential are discussed, including heart failure, infarction, arrhythmias, syncope, cardiomyopathy, angina, heart transplantation and coronary bypass grafts, coronary artery disease, diabetic cardiomyopathy, hypertension, ischemia, thrombosis, diabetes mellitus, and migraine., (© 2017 American Heart Association, Inc.)

P2X receptors on dorsal root ganglion (DRG) neurons have been strongly implicated in pathological nociception after peripheral nerve injuries or inflammation. However, nothing is known of a role for purinergic receptors in neuropathic pain produced by a chronic compression of DRG (CCD) - an injury that may accompany an intraforaminal stenosis, a laterally herniated disc or other disorders of the spine leading to radicular pain. In a rat model of DRG compression, hyperexcitable neurons retain functioning axonal connections with their peripheral targets. It is unknown whether such hyperexcitability might enhance chemically mediated nociceptive stimulation of the skin. In this study, CCD facilitated the nocifensive behavior and mechanical hyperalgesia-induced by the P2X 3 agonist, alpha,beta-methylene ATP (alpha,beta-meATP). An injection of alpha,beta-meATP into the hind paw of CCD rats resulted in a significantly greater decrease in the mean threshold to von Frey stimuli and a greater duration of paw lifts than in sham-operated control rats. CCD also increased the levels of P2X 3 receptor protein and the number of P2X 3 immunoreactive, small diameter DRG neurons in the compressed ganglion. P2X 3 receptors were co-labeled with the isolectin IB4, consistent with a role in nociception. In addition, a alpha,beta-meATP induced significantly larger fast-inactivating currents in CCD- than in sham-operated acutely dissociated DRG neurons. These currents were accompanied by the generation of action potentials - but only in the CCD neurons. U0126, a specific inhibitor of the MEK1/2, greatly down-regulated the enhanced current. Taken together, these observations suggest that enhanced purinergic responses after CCD are mediated by P2X 3 receptors.

Objectives: Extracellular adenosine 5'-triphosphate modulates the functions of the adult pancreas via 2 nucleotide receptor families, the P2X and P2Y receptors. Expression of the P2X7 receptor has been demonstrated in islet cells of the pancreas, particularly the mature alpha cells that secrete glucagon. In the streptozotocin-induced diabetic model, a loss of insulin-secreting cells was accompanied by an increase in alpha cells that expressed the P2X7 receptor., Methods: In the present study, we have examined the expression of P2X7 receptors in the developing pancreas from embryonic days 10 (E10) to E18., Results: We detected P2X7 receptor-immunoreactive cells in pancreatic islet cells as early as E11' before glucagon expression. Subsequently, P2X7 receptors were expressed in glucagon-secreting cells at E12, and complete colocalization was observed at E14. Occasional colocalization of P2X7 receptors and insulin was observed in scattered cells at E12 and E14, but not at E18, when the glucagon- and insulin-secreting cells were almost completely segregated., Conclusions: It was found that P2X7 receptors were expressed early in a subpopulation of glucagon- and insulin-immunopositive cells in developing islets and subsequently became restricted to glucagon-expressing cells as development proceeded. The possible functional significance of these changes is discussed.

In this study, we have used reverse transcriptase polymerase chain reaction and immunocytochemistry to show that P2X1, P2X2, P2X4, P2X5, P2X6 and P2X7 receptor messenger RNA and protein are expressed in the rat choroid plexus taken from the fourth and lateral ventricles. In some epithelial cells, the apical surfaces were stained more intensely than the basal surfaces. Some of the epithelial cells showed strong staining, others weak or no staining. Double immunostaining with P2X and cytokeratin antibodies or isolectin B4 binding confirmed that P2X receptors were localized in subpopulations of the choroid plexus epithelial cells, rather than the endothelial cells of capillaries. The results suggest that P2X receptors might be involved in the regulation of cerebrospinal fluid composition.

Objective: To investigate the interaction of exogenous adenosine 5'-triphosphate (ATP), a P2 receptor agonist, with prostaglandin F(2alpha) (PGF(2alpha)) on pregnant women in labor as well as on isolated human pregnant uterus preparations., Methods: For an in vitro study, myometrial samples were obtained from 27 women undergoing elective cesarean delivery at term. Concentration-response relationships for ATP (10(-8) -3 x 10(-4) mol/L), PGF(2alpha) (10(-9) -10(-5) mol/L), and their combination were obtained by using routine pharmacological organ bath technique. An in vivo study was performed with 34 pregnant women with dysfunctional abnormalities of the active stage of labor who were randomly allocated into 2 study groups. The women in the control group (18 patients) received intravenous prostaglandin F(2alpha) at an initial rate of 7.5 mug/min, whereas the women in the ATP group (16 patients) received prostaglandin F(2alpha) concomitantly with ATP (0.45 nmol/min, intravenously)., Results: Adenosine 5'-triphosphate at concentrations of 10(-6) -3 x 10(-4) mol/L and PGF(2alpha) at concentrations of 10(-8) -10(-5) mol/L caused concentration-dependent contractions of isolated smooth muscle preparations of the human pregnant uterus. At concentrations of 10(-6) mol/L and below, ATP had no effects on mechanical activity of the isolated uterus, but at concentrations of 10(-7) mol/L and 10(-6) mol/L, it significantly potentiated the contractile responses of the uterus induced by PGF(2alpha) (P < .05, 2-way analysis of variance). Patients receiving intravenous infusion of ATP as a supplement to PGF(2alpha) treatment, compared with those without ATP, had a significantly shorter interval from the start of the treatment to full cervical dilatation (3.31 +/- 1.49 hours and 4.67 +/- 1.11 hours in ATP and control groups, respectively; P = .014, Wilcoxon Mann-Whitney test). The total dose of prostaglandin received was significantly lower in the ATP group than that of controls (1,489.8 +/- 699.9 mug and 3,394.2 +/- 1,951.9 mug, respectively; P = .003, Wilcoxon Mann-Whitney test). No side effects of ATP treatment were observed during or after infusion., Conclusion: Adenosine 5'-triphosphate potentiates effects of PGF(2alpha) on pregnant human uterus in vitro and in vivo and thus could be a useful supplemental drug to increase uterine contractility at labor.

Evidence for the role of purinergic signaling (via P1 and P2Y receptors) in the proliferation of vascular smooth muscle and endothelial cells is reviewed. The involvement of the mitogen-activated protein kinase second-messenger cascade in this action is clearly implicated, although details of the precise intracellular pathways involved still remain to be determined. Synergistic actions of purines and pyrimidines with growth factors occur in promoting cell proliferation. Interaction between purinergic signaling for vascular cell proliferation and cell death mediated by P2X7 receptors is discussed. There is evidence of the release of ATP from endothelial cells, platelets, and sympathetic nerves as well as from damaged cells in atherosclerosis, hypertension, restenosis, and ischemia; furthermore, there is evidence that vascular smooth muscle and endothelial cells proliferate in these pathological conditions. Thus, the involvement of ATP and its breakdown product, adenosine, is implicated; it is hoped that with the development of selective P1 (A2) and P2Y receptor agonists and antagonists, new therapeutic strategies will be explored.

In response to increased shear stress, vascular endothelial cells release adenosine triphosphate (ATP) by an unknown mechanism. We have investigated this mechanism using different approaches. First, we discovered that quinacrine, used to locate intracellular stores of ATP bound to peptides, displayed a granular fluorescence, typical of vesicular storage. Second, we found that two inhibitors of vesicular transport (monensin and N-ethylmaleimide) produced a highly significant reduction in the release of ATP from vascular endothelial cells in response to increased shear stress. Preliminary experiments using inhibitors of the cystic fibrosis transmembrane regulator, the sulfonylurea receptor, and the multidrug resistance protein showed no involvement of these ATP-binding cassette transporter proteins (previously characterized in endothelial cells) in the mechanism of release of ATP. We suggest, therefore, that the release of ATP from vascular endothelial cells, like that of nerve cells, is probably by vesicular exocytosis.

The pattern of distribution and co-localization of nitric oxide synthase (NOS) and quinacrine fluorescence (indicative of vesicular adenosine 5'-triphosphate, ATP), and co-localization of NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity in the myenteric plexus of pre-term human fetal (6-17 weeks of gestation) stomach and small intestine was examined using immunohistochemical and histochemical techniques. In all stages of gestation investigated, NOS-immunoreactive and NADPH-d-reactive myenteric neurons and nerve fibres were seen in the fetal intestine and stomach. However, in fetuses of 6-10 weeks of gestation, only 15% of the NADPH-d-positive myenteric neurons were NOS-immunoreactive, whereas a 100% co-localization was found in samples of 12-17 weeks of gestation. Quinacrine fluorescent myenteric neurons and nerve fibres were found only in the fetal intestine of 12-17 weeks of gestation, of which 25% of the NADPH-d-positive myenteric neurons in these samples were quinacrine fluorescent. These findings demonstrate the presence and co-localization of markers for nitric oxide (NO)- and ATP-utilizing myenteric neurons and nerve fibres in the early stages of gestation, suggesting possible co-transmitter and/or trophic roles of ATP and NO in the process of development and maturity of human myenteric neurons. In addition, the fact that only a small percentage of NADPH-d-reactive myenteric neurons express NOS immunoreactivity at 6-10 weeks of gestation confirms that NADPH-d-reactivity does not always represent NOS activity.

P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.

The endothelium is recognized as a major source of endothelin in the vasculature. In this report we show for the first time that endothelin can be demonstrated with immunoelectronmicroscopy in perivascular nerves. About 36% of the axon profiles (varicosity/intervaricosity) examined in the rat basilar artery showed immunolabelling with a polyclonal antibody to endothelin-1. Endothelin-labelled axon varicosities were characterized by the presence of small spherical agranular vesicles (42 +/- 1 nm); some varicosities also contained large granular vesicles (92 +/- 5 nm) with labelled cores. Immunolabelling was mostly distributed in the axoplasm and in association with the membrane of synaptic vesicles (the lumen of the small agranular vesicles was immuno-negative). The presence of endothelin in perivascular nerves of the basilar artery suggests a neuronal as well as endothelial role in the physiological control of the vessel wall.

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.

Pharmacological studies have demonstrated that various purinoceptors are involved in the control of the cerebral vascular tone in many species. In this study, the existence of P2X purinoceptors in the postmortem human cerebral arteries was investigated with organ-bath pharmacology, autoradiography, and immunohistochemistry. Specimens were obtained from the M2 region of the middle cerebral arteries from human cadavers with an age range of 53-91 years and postmortem time of 37-54 h. Application of alpha,beta-methylene adenosine triphosphate (ATP) produced concentration-dependent contraction in the arterial ring, whereas transmural nerve stimulation and noradrenaline did not elicit contraction. Autoradiography using [3H]alpha,beta-methylene ATP (a radioligand for P2X purinoceptors) showed specific [3H]alpha,beta-methylene ATP binding sites in the smooth-muscle cells of the postmortem human cerebral arteries. Immunohistochemistry with specific P2X1 purinoceptor antibodies revealed positive staining exclusively in the smooth muscle of the same specimens. All these results demonstrate the existence of P2X purinoceptors in human cerebral arteries, which were still functionally active despite the long postmortem time. The results from this study suggest that the postmortem human cerebral arteries can be useful specimens for studying the P2X purinoceptor-mediated responses.

The sensory neurone-specific ATP-gated cation channel P2X3, when expressed in Xenopus oocytes, desensitizes rapidly. Complete removal of extracellular calcium abolishes desensitization. Pretreatment of oocytes with cyclosporin also abolishes P2X3 desensitization. When the calcineurin auto-inhibitory peptide (CaN A457-481) was injected into oocytes, the rate of desensitization of P2X3 decreased on consecutive applications of ATP, suggesting a role for calcineurin in the regulation of desensitization. Truncated P2X3 clones initiating at Met-75 lack the N-terminal intracellular region, but express functional channels in oocytes that do not desensitize. Taken together, these data suggest that P2X3 desensitizes through a calcium-dependent calcineurin-mediated dephosphorylation involving N-terminal residues that are phosphorylated in functional channels.

Recent pharmacological studies have shown that perivascular nerves can influence the development and function of vascular endothelial cells (ECs). However, morphological studies have not yet been carried out to investigate whether these functional changes are associated with changes in vasoactive substances in ECs. We used postembedding electron microscopy (EM) triple gold-labeling immunocytochemistry to study the effects of short-term sympathectomy (3 days after 6-hydroxydopamine [6-OHDA] treatment) and long-term sympathectomy (guanethidine and 8 days after 6-OHDA) on the distribution of vasoactive substances in ECs of the rat thoracic aorta. The post-embedding immunocytochemistry, which can detect levels of label in individual cells, showed that there was a significant decrease in endothelial NO synthase (NOS3)-labeled, serotonin (5-HT)-labeled, and substance P (SP)-labeled, but a significant increase in endothelin-1 (ET-1)-labeled, gold particles in ECs after long-term, but not after short-term (3-day), sympathectomy. In conclusion, our results show that long-term sympathectomy causes an increase in ET-1 and decrease in NOS3, 5-HT, and SP immunoreactivity in ECs of the thoracic aorta. Our data also indicate that postembedding EM triple gold-labeling immunocytochemistry is a valuable technique for quantitative studies of the content of vasoactive substances in ECs.

We investigated the effects of several concentrations of extracellular ATP on the release of intracellular ATP by human umbilical vein endothelial cells (HUVEC) in primary cultures. When ATP is added to the medium of cultured EC at a concentration of 1 microM, it is readily degraded by extracellular enzymes; 10 microM ATP added to the culture medium provokes a transient but significant increase, followed by a decrease in the concentration of extracellular ATP. At a concentration of 100 microM, there was a significant release of ATP and its level was maintained in the culture medium throughout the experiment. Our results show that extracellular ATP leads to a sustained release of intracellular ATP by HUVEC. Such sustained self-perpetuating release of ATP is likely to play an important part in physiological and pathological local vascular control mechanisms.

In previous studies, we have demonstrated that enteric ganglia can survive when transplanted into the striatum. However, if such grafts are to be effective in clinically significant situations, it is necessary for them to survive in the brain for long periods. In this study, we have examined the corpus striatum of host rats one year after transplantation of pieces of myenteric plexus taken from young Fischer rats. NADPH-diaphorase-containing enteric neurones had survived within the CNS environment one year after grafting, and had extended axons into the surrounding striatum.

The effect of removal of the pituitary on mesenteric arterial function was examined in adult male rats that had undergone hypophysectomy 7 days earlier. Sham-operated rats and weight-matched rats served as controls. The body weight of hypophysectomized rats decreased from 220.75 +/- 0.48 g to 188.03 +/- 2.53 g (n = 7). Sham-operated controls gained weight, from 223 +/- 1.47 to 275.85 +/- 3.45 g (n = 8). Frequency-dependent vasoconstriction to electrical field stimulation (2-32 Hz, 90 V, 1 ms, 30 s) was significantly augmented after hypophysectomy. The maximal constrictor response of hypophysectomized preparations, 215.5 +/- 14.9 mm Hg (n = 6), was approximately twice that of the sham-operated controls, 100 +/- 6.1 mm Hg (n = 7) and weight-matched controls 109.8 +/- 5.8 mm Hg (n = 8). Norepinephrine (NE) (0.05-1,500 nmol) elicited dose-dependent vasoconstriction; the maximal response was significantly augmented after hypophysectomy, 221.71 +/- 15.9 (n = 7) as compared with 148.0 +/- 16.0 mm Hg (n = 8) in sham-operated controls and 146.3 +/- 8.7 mm Hg (n = 7) in weight-matched controls. Dose-dependent vasoconstrictor responses to ATP, 5-hydroxytryptamine, vasopressin, and endothelin were similar between the groups. High-performance liquid chromatography analysis showed no difference in NE content of the superior mesenteric artery from hypophysectomized and sham-operated controls. Hypophysectomy of rats caused an increase in sympathetic constrictor function of the mesenteric arterial vasculature that appeared to involve postjunctional adrenoceptors rather than prejunctional mechanisms and was not due to arrested growth or the smaller size of the mesenteric preparations.

We reported previously that myenteric plexus explants promoted striatal neurite elongation in co-culture and that this effect was abolished by tetrodotoxin (TTX). Here we demonstrate that the nitric oxide synthase blocker N-nitro-L-arginine methyl ester significantly reduced the neuritogenic effect of the myenteric plexus whereas the nitric oxide donor, sodium nitroprusside (SNP), partially reversed the blocking effect of TTX. 2-Chloroadenosine (2-CA), a stable analogue of adenosine, which is produced following release of ATP from enteric neurones, further enhanced the effect of SNP. Basic fibroblast growth factor or neurotrophin-3 in combination with 2-CA and SNP were only marginally neuritogenic in striatal cultures alone. These results suggest that NO is involved in the trophic effects of myenteric plexus explants on striatal neurones.

The effects of the stable adenosine analogue, 2-chloroadenosine (2-CA) and basic fibroblast growth factor (bFGF) on myenteric neurones in dissociated cell culture were examined. 2-CA had no effect on neuronal numbers, but increased neurite length, in a dose-dependent manner. bFGF increased both the number of myenteric neurones and neurite length. When 2-CA was applied together with bFGF, an enhanced increase in neurite outgrowth, but no additional increase in neuronal numbers was observed. 2-CA-induced effects were blocked by the adenosine antagonist 8-(p-sulphophenyl)theophylline. These results show, for the first time, that both purines and bFGF may have trophic actions on myenteric neurones and also indicate that purines enhance some effects of bFGF, in a synergistic manner.

Objective: To investigate the possible role of substance P as an endothelial factor in the local regulation of vascular tone in the human ovarian vein., Methods: We performed immunolocalization of substance P in human ovarian venous endothelium in situ and in culture, and observed responses to substance P in preconstricted ring preparations of human ovarian vein in the presence of either the prostaglandin synthesis inhibitor indomethacin or the inhibitor of nitric oxide synthesis L-nitro arginine methyl ester (L-NAME), with and without luminal rubbing., Results: Substance P was localized in a subpopulation of ovarian vein endothelial cells. Maximal relaxation induced by substance P was not significantly affected by indomethacin (10 mumol/L), but was reduced from 58.7% (95% confidence interval [CI] 41.3-76.1) in control experiments to 24.7% (95% CI 18.3-31.1) after luminal rubbing and to 32.3% (95% CI 19.8-44.8) after exposure to L-NAME (0.1 mmol/L) (P = .001)., Conclusion: The localization of substance P in ovarian vein endothelium together with vasodilator effects mediated partially via the endothelium suggests that the peptide has a role in the local control of vascular tone in this vessel.

The distribution of [3H]alpha,beta-methylene ATP ([3H]alpha,beta-MeATP) binding sites in rat brain and spinal cord was examined using autoradiography. It was shown that many structures in the CNS are densely labelled, and that the binding is displaced by both beta,gamma-methylene ATP and the P2X-purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), indicating that the binding sites are P2X-purinoceptors. Image analysis revealed that the nuclei of the thalamus have the highest density of labelling. Other densely labelled structures include: the amygdaloid, substantia nigra, cerebral cortex, hypothalamus, caudate putamen, geniculate nuclei, medial habenula and the intermediate zone of grey matter in the spinal cord. These results are consistent with the limited amount of electrophysiological evidence for the existence of P2X-purinoceptors in the CNS, and provoke the need for extensive physiological studies to establish the roles of P2X-purinoceptors in the CNS.

The Langendorff heart preparation was used to investigate the mechanism of action of vasodilatation evoked by ATP and its analogues in guinea pig coronary vasculature. The relative order of potency of ATP and its analogues in causing a reduction in perfusion pressure was 2-methylthioATP (2-meSATP) > ATP > beta, gamma-methyleneATP (beta,gamma-meATP) > or = alpha,beta-methyleneATP (alpha,beta-meATP), thus establishing the presence of P2y-purinoceptors in this preparation. L-NG-nitroarginine methyl ester (L-NAME, 3 x 10(-5) M) significantly attenuated both the area under the flow-time curve and the maximum decrease in perfusion pressure of the vasodilatation produced in response to 2-meSATP (5 x 10(-12)-5 x 10(-9) mol). However, for ATP (5 x 10(-7)-5 x 10(-10) mol), L-NAME 3 x 10(-5) M significantly attenuated the area under the flow-time curve of the response but did not reduce the maximum decrease in perfusion pressure except at one low dose (5 x 10(-10) mol). L-Arginine 1.5 x 10(-3) M significantly reversed inhibition of the area under the flow-time curve of the response to 2-meSATP 5 x 10(-10) mol and ATP 5 x 10(-8) mol by L-NAME 3 x 10(-5) M. The maximum decrease in perfusion pressure of the response to ATP 5 x 10(-10)-5 x 10(-7) mol was significantly attenuated in the presence of indomethacin 10(-6) M.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied purinoceptor subtypes mediating constriction and dilatation in isolated rabbit coronary arteries with and without endothelium by comparing the effects of adenosine, ATP and the ATP analogues, alpha, beta-methylene ATP and 2-methylthio ATP. For contraction, the rank order of agonist potency was alpha, beta-methylene ATP > 2-methylthio ATP >> ATP; adenosine did not produce contraction. Removal of the endothelium did not affect these responses, but they were abolished by previous desensitisation with alpha, beta-methylene ATP. For relaxation, adenosine, ATP, and 2-methylthio ATP were equipotent, but alpha, beta-methylene ATP had no vasodilator effect and caused further contraction of the preparations. A transient contraction often preceded the relaxations to ATP at higher concentrations. The dilator responses to adenosine, ATP, and 2-methylthio ATP were significantly reduced but not abolished in preparations denuded of endothelium; previous desensitisation with alpha, beta-methylene ATP had no effect on the relaxant responses but abolished the initial transient contractions to ATP and 2-methylthio ATP. These results indicate the presence of vasodilator P1- and P2Y-purinoceptors on smooth muscle and, to a lesser extent, on endothelium and P2X-purinoceptors, mediating transient vasoconstriction on smooth muscle alone. The presence of smooth muscle P2Y-purinoceptors is unusual and has functional implications regarding the nature of the response of the coronary artery to ATP.

The effect of streptozotocin-induced diabetes on the production of vasoactive intestinal polypeptide messenger RNA in the myenteric neurones of 8 weeks diabetic rat intestine was examined using in situ hybridization. Total ganglion cell number per length (mm) of tissue section was measured using NADH diaphorase histochemistry. Although ganglion cells were more numerous in the control preparations compared with diabetic samples, a significantly greater number of vasoactive intestinal polypeptide messenger RNA-containing cells was detected in the diabetic tissues. These observations suggest that there is either a decrease in the breakdown of vasoactive intestinal polypeptide messenger RNA or an increase in its synthesis in myenteric neurones of diabetic rat intestine.

Pharmacological evidence suggests that nitric oxide (NO) is involved in non-adrenergic, non-cholinergic, nerve mediated responses seen in guinea-pig trachealis muscle. The synthetic enzyme for NO (NO synthase) has recently been shown to be responsible for neuronal NADPH-diaphorase activity. Therefore, to determine whether intrinsic paratracheal neurones could be a source of NO in the trachea, expression of NADPH-diaphorase activity was examined histochemically using whole mount preparations of the tracheal plexus. Many paratracheal neurones were found to express moderate to high levels of NADPH-diaphorase activity and are thus likely to be a source of NO in this tissue. This observation provides further evidence that NO is involved in the regulation of relaxation in airway smooth muscle.

Age-related changes in the expression of galanin- and calretinin-like immunoreactivity in the ganglionated plexus of 2-4 day, 6 month and 2 year old guinea-pig gallbladder were investigated. The ganglionated plexus was studied using immunohistochemical labelling techniques on whole mount stretch preparations of the gallbladders. Galanin- and calretinin-like immunoreactivity did not differ significantly between age groups of the 2-4 day and 6 month olds except for some reduction of calretinin-immunofluorescent nerves in the 6 month old preparations. However, in the ganglionated plexus of the 2 year old guinea-pig gallbladders, neither galanin- nor calretinin-like immunoreactivity could be detected. The implication of these findings, in relation to the physiological activity of the gallbladder is discussed.

The distribution and colocalization of nitric oxide synthase (NOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase was studied in the neuronal elements of the adrenal gland of the rat. Ganglion cells and many nerve fibres in the gland showed both NOS-immunoreactivity and NADPH-diaphorase staining. The adrenal cortical cells showed NADPH-diaphorase staining but were not immunoreactive for NOS. Positive labelling for both NADPH-diaphorase and NOS was found in bundles and in single fibres with varicosities, preferentially located around the noradrenaline (NA)-storing cells. Adrenaline (A)-storing cells and ganglion cells in the medulla, along with the cortical cells and blood vessels in the zona glomerulosa, received relatively fewer positive fibres.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

The responses to vasoactive agents and the fine structure of hepatic arterial ring segments from male and female Watanabe heritable hyperlipidaemic (WHHL) rabbits (4, 6, and 12 months) were compared with those of age- and sex-matched New Zealand White (NZW) rabbits. In males only, KCl-induced contractions were reduced in WHHL rabbits compared with NZW rabbits. In male and female WHHL rabbits, maximum noradrenaline-induced contractions and sensitivity to noradrenaline were greater than those of male and female NZW rabbits. In female WHHL and NZW rabbits only, maximum noradrenaline-induced contractions and the EC50 values were reduced at 6 months. Endothelium-dependent relaxation: In females only, maximum relaxant responses and the sensitivity of WHHL rabbits to acetylcholine increased with age, while there was a decrease in NZW rabbits. Similarly, relaxation to substance P increased with age in WHHL rabbits and decreased in NZW rabbits, but this occurred in both male and female animals. In addition, substance P-induced relaxation in female WHHL rabbits was greater than in male WHHL rabbits. Endothelium-independent relaxation: In both male and female WHHL rabbits, calcitonin gene-related peptide-induced and vasoactive intestinal polypeptide-induced relaxation did not change with age. However, there was an age-related decrease in the response of NZW rabbits to these peptides. Electron microscopic evaluation of hepatic arteries from WHHL rabbits showed occasional ruptures in the internal elastic lamina at 4 months. At 6 months, widespread intimal thickening associated with smooth muscle cell migration was apparent, but this became less obvious at 12 months. No obvious differences in structure between male and female hepatic arteries were observed. It is suggested that a "compensatory vasodilatation" develops in atherosclerosis, initially at the level of the endothelium, and then with the progression of the disease extends to changes in the smooth muscle. This may occur in order to offset the thickening of the arterial wall. Sexual dimorphism in vascular reactivity has been demonstrated.

The effects of the purines, adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP), and the pyrimidines, uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate (CTP), and thymidine 5'-triphosphate (TTP), on vascular resistance were investigated in the rat mesenteric arterial bed. In preparations at basal tone, these agents produced dose-related vasoconstriction with a potency order of ATP greater than CTP greater than UTP much greater than TTP = GTP. When tone was raised with norepinephrine (30 microM), these agents caused dose-related vasodilatation with the potency order of UTP = ATP greater than TTP = GTP. CTP did not elicit vasodilatation. Removal of the endothelium with sodium deoxycholate resulted in an increased responsiveness of the mesenteric bed preparation to the vasoconstrictor effects of each of the purines and pyrimidines tested. The selective P2 X-purinoceptor-desensitizing agent alpha,beta-methylene ATP inhibited vasoconstrictor responses to ATP and to CTP but had no effect on vasoconstrictor responses elicited by UTP, TTP, and GTP. In raised-tone preparations, vasodilator responses to ATP, UTP, TTP, and GTP were abolished after removal of the endothelium with sodium deoxycholate. Responses to acetylcholine were also abolished; those to sodium nitroprusside were unimpaired. An inhibitor of the formation of nitric oxide from L-arginine, N omega-nitro-L-arginine methyl ester (30 microM), which antagonizes responses mediated by endothelium-derived relaxing factor (nitric oxide), attenuated vasodilatation to ATP, UTP, and acetylcholine but not to sodium nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of P2-purinoceptor subtypes has facilitated understanding of the many diverse effects produced by purine nucleotides. P2X-Purinoceptors are located on vascular smooth muscle where they mediate vasoconstriction resulting from ATP released as a cotransmitter with noradrenaline from sympathetic nerves. P2Y-Purinoceptors are usually located on the vascular endothelium where they have a role as mediators of vascular relaxation by locally produced ATP. In some vessels, P2Y-purinoceptors are also located on the smooth muscle, perhaps in association with purinergic or sensory nerves, where they can elicit direct relaxation to neuronally released ATP. The net effect of ATP and its analogues on isolated vessels or on vascular beds will be the results of actions mediated by P2X- and P2Y-purinoceptor subtypes, although changes in vascular tone and in integrity of nerves and endothelial cells may alter the balance of the response. Such changes have been observed in diseased states (e.g., atherosclerosis) and may have important implications for the involvement of P2-purinoceptors in, for example, vasospasm. The development of selective and potent antagonists to P2X- and P2Y-purinoceptors has so far remained elusive, and their therapeutic potential can only be guessed.

Vasomotor responses of isolated coronary arteries to peptide and nonpeptide neurotransmitter agents changed with the development of sexual maturity in female New Zealand white rabbits aged 4-12 months. There was a significant reduction from 4- to 12-month-old animals in both the direct smooth muscle vasodilator responses to calcitonin gene-related peptide (p less than 0.01) and vasoactive intestinal polypeptide (p less than 0.001), and in the endothelium-mediated response to substance P (p less than 0.005). Vasodilator responses to concentrations of acetylcholine (ACh) greater than 0.1 microM were virtually absent in the 6- and 12-month-old animals. No change in maximal relaxation to noradrenaline was seen with maturation, although there was a small significant increase in potency. The contractile response of the smooth muscle to 30 mM KCl declined steadily as the animals matured, but the maximal contraction to ACh (p less than 0.05), neuropeptide Y (p less than 0.02), and serotonin (p less than 0.05) increased significantly between 4 and 12 months of age. These results indicate that following sexual maturation in the female rabbit, the epicardial coronary artery shows a significant increase in maximum responses to vasoconstrictor neurotransmitter agents and, at the same time, a marked decline in responses to some vasodilator agents, both those acting directly on the smooth muscle and those acting via the endothelium.

In addition to the classical transmitters noradrenaline and acetylcholine, other transmitters have been identified in perivascular nerves, including 5-hydroxytryptamine, ATP and a number of peptides. This paper discusses pre- and postjunctional neuromodulation of vascular transmission, and cotransmission involving noradrenaline, ATP and neuropeptide Y in sympathetic nerves, acetylcholine and vasoactive intestinal polypeptide in parasympathetic nerves, and substance P, calcitonin gene-related peptide and ATP in 'sensory-motor' nerves. Vasomotor nerves derived from intrinsic neurones, for example in the heart and gut, are also discussed. Subpopulations of endothelial cells store and release a variety of substances, including acetylcholine, substance P, ATP, 5-hydroxytryptamine, vasopressin and angiotensin II, that act on receptors on endothelial cells and lead to the production of endothelium-derived relaxing factor (identified as nitric oxide) which, in turn, produces vasodilation in response to changes in flow and hypoxia. Endothelium-derived contracting factors such as endothelin may also be released. There appears to be a resting dynamic balance between endothelium-derived vasodilator tone and sympathetic vasoconstrictor tone, which is altered under different physiological and pathophysiological circumstances. Long-term (trophic) interactions between perivascular nerves and endothelial cells are discussed, as are the changes in vascular control mechanisms that occur with ageing and hypertension and in the nerves that remain following trauma or surgery.

We have examined the neuropeptide Y-containing intrinsic nerves of the heart in young (6-week-old) and adult (4-month-old) rats to determine whether they project to the coronary arteries or are capable of doing so if the neuropeptide Y-containing extrinsic nerves are removed. Chronic treatment of neonates with guanethidine was used to permanently destroy the sympathetic nerves. In the young treated animals, 33-54% of the neuropeptide Y remained in the heart despite a 90-99% reduction in norepinephrine; these proportions did not change in the animals that were allowed to develop to adulthood. The level of neuropeptide Y in the right atrium of young animals was unexpectedly high (252 +/- 28.7 pmol/g) compared with adults (75.4 +/- 18.8 pmol/g). The coronary arteries in the control rats received a moderately dense supply of neuropeptide Y-containing nerves; after guanethidine, all neuropeptide Y-containing nerves innervating the large coronary arteries disappeared, but some were still seen in association with small resistance vessels. No compensatory proliferation of the intrinsic neuropeptide Y-containing neurons occurred in the adult sympathectomized animals, and the intrinsic nerves did not reinnervate the large coronary arteries. These results are discussed in relation to the clinical syndrome of coronary artery spasm.

The rat hind-limb vasculature releases substance P when subjected to a rapid increase in flow through the vascular bed. This release also occurs during high flow after rats have been capsaicinized, when loss of substance P-containing nerve fibers was verified by immunohistochemistry. Air treatment, a procedure shown by transmission electron microscopy to have removed endothelial cells from the arteries but not arterioles or capillaries of the hind-limb preparations, eliminated this release. Thus, the substance P released is unlikely to arise from perivascular nerves but rather from arterial endothelial cells.

A wide variety of substances are utilized as neurotransmitters in perivascular autonomic nerves, including noradrenaline, acetylcholine, ATP, 5-hydroxytryptamine, and a number of peptides. A general model for the autonomic innervation of the vessel wall is outlined, and variations in the innervation patterns in different precapillary vessels are described. Evidence that noradrenaline and ATP are cotransmitters in some precapillary vessels is presented. In the rat tail artery, ATP appears to be responsible for the fast depolarizations that are recorded in response to perivascular nerve stimulation, whereas noradrenaline is responsible for the slow maintained depolarization.

In 10 patients with chronic autonomic failure, the sympathetic perivascular nerve plexuses from quadriceps muscle biopsies were studied by catecholamine fluorescence and electronmicroscopy. There was almost complete absence of catecholamine fluorescence and fewer than normal numbers of small granular (noradrenergic) vesicles in all nerves studied. The most marked depletion of noradrenergic vesicles was seen in two of the patients with pure autonomic failure, but more studies are needed for a full quantitative comparison of pure autonomic failure and autonomic failure with multiple system atrophy (Shy-Drager syndrome).

A dual function for adenosine 5'-triphosphate in the regulation of vascular tone is considered. Adenosine 5'-triphosphate can cause vasodilation, acting via P2-purinoceptors located on vascular endothelial cells to release an endothelium-derived relaxing factor which diffuses to the vascular smooth muscle and induces vasodilation. The main source of intraluminal adenosine 5'-triphosphate is likely to be endothelial cells, and its release can be measured during pathophysiological conditions such as ischemia and hypoxia, in amounts likely to be sufficient to activate endothelial P2-purinoceptors. Adenosine 5'-triphosphate can also be released during intravascular platelet aggregation and from intact and damaged vascular smooth muscle cells, and so may play a role in the complex physiological mechanisms controlling local vascular tone under normoxic conditions and during vessel injury. Evidence is also presented for adenosine 5'-triphosphate acting as an excitatory cotransmitter with noradrenaline from sympathetic perivascular nerves, to cause vasoconstriction via excitatory P2-purinoceptors located on vascular smooth muscle. The postjunctional mechanical and electrical responses of a number of blood vessels following perivascular nerve stimulation contain a component that is resistant to blockade of the alpha-adrenoceptor. This nonadrenergic response is mimicked by adenosine 5'-triphosphate and can be blocked by selective desensitization of the P2-purinoceptor by alpha,beta-methylene adenosine 5'-triphosphate. Vesicular storage of adenosine 5'-triphosphate and its release from sympathetic perivascular nerves has also been demonstrated. The functional significance of adenosine 5'-triphosphate acting intraluminally as a vasodilator and extraluminally as a vasoconstrictor neuronal agent in the control of vascular tone is discussed.

The recent discoveries of vascular neuroeffector control mechanisms, involving a wide variety of neurohumoral agents, pre- and postjunctional neuromodulation, and cotransmission, leave the field poised for growth in new directions. Some of these are outlined in this article, including: the development of methods for quantitation of the pattern and density of different types of perivascular nerves; exploration of the potent actions of purine nucleotides and nucleosides on vascular smooth muscle and/or endothelial cells, particularly in relation to the development of drugs of therapeutic potential; expansion of studies of the regulatory implications of cotransmitter release of ATP together with noradrenaline from some sympathetic perivascular nerves and of VIP together with acetylcholine from some parasympathetic nerves; autoradiographic localization of receptors for monoamines, peptides of and purines in blood vessels; wider studies of "axon reflex" control of the circulation and of the roles of substance P; investigation of development, aging, and regeneration of different perivascular nerve types, and the long-term "trophic" actions of some neurohumoral agents. Lastly, the time is ripe to study abnormalities in neurohumoral control of vessels in disease and after chronic exposure to drugs.

A new protocol has been developed for selective removal of the endothelium from the arteries of the perfused rat hind-limb preparation. The hind limb was perfused with oxygenated Krebs-Ringer bicarbonate solution with a combination of high flow and timed air bubbles (2 minutes at high flow followed by 5 minutes of air bubbles, followed by a further 2 minutes at high flow). Representative vessels at different diameters of the arterial tree--the femoral artery, the artery supplying the extensor hallucis proprius muscle, and the arteries, arterioles, and capillaries within the hallucis proprius muscle--were taken for examination by transmission and scanning electron microscopy. A graded degree of damage occurred along the length of the hind-limb vasculature, with endothelial cells having been completely removed from the femoral artery, removed from or severely damaged in the artery supplying the hallucis proprius muscle, and partially damaged in arteries within the hallucis proprius muscle. However, no damage occurred to the endothelial cells of the arterioles and capillaries within the hallucis proprius muscle. The integrity of the vascular bed after endothelial removal according to the above protocol was confirmed by the demonstration of no diminution, but in fact an increase, in contractile responses to bolus injections of alpha,beta-methylene ATP, resulting in a shift to the left of the dose-response curve. The viability was further confirmed by the fact that the conductance (flow/perfusion pressure) of the preparation during the periods of high flow was no different before and after removal of the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

The vascular neuromuscular junction is described and the terms neuromodulation and cotransmission defined. Two main types of interaction between peptide and nonpeptide neurotransmitter substances during the local control of blood flow are distinguished. The first concerns the interacting roles of peptides and nonpeptides that coexist in, and are released from, perivascular nerve varicosities. Examples include the interactions of neuropeptide Y (NPY) with noradrenaline (NA) and adenosine 5'-triphosphate (ATP) released from some sympathetic nerves; vasoactive intestinal polypeptide (VIP) with acetylcholine (ACh) released from some parasympathetic nerves; and NPY and 5-hydroxytryptamine (5-HT) released from intracardiac neurones supplying coronary vessels. Possible interactions of substance P (SP) and calcitonin gene-related peptide (CGRP) with ATP released from some primary afferent sensory nerves are also considered. The second type of interaction concerns vascular neurotransmitters and locally released agents such as histamine, prostanoids, and bradykinin. Finally, a hypothesis is put forward in which it is suggested that peptides and nonpeptides released from endothelial cells during hypoxia lead to protective vasodilatation via receptors on the endothelium, while these substances released from perivascular nerves during different physiological circumstances usually constrict the blood vessel via receptors on the smooth muscle cells.