Neuropeptide Y: Direct vasoconstrictor and facilitatory effects on P2X1 receptor-dependent vasoconstriction in human small abdominal arteries

Neuropeptide Y (NPY) is co-released with norepinephrine and ATP by sympathetic nerves innervating arteries. Circulating NPY is elevated during exercise and cardiovascular disease, though information regarding the vasomotor function of NPY in human blood vessels is limited. Wire myography revealed NPY directly stimulated vasoconstriction (EC50 10.3 ± 0.4 nM; N = 5) in human small abdominal arteries. Maximum vasoconstriction was antagonised by both BIBO03304 (60.7 ± 6%; N = 6) and BIIE0246 (54.6 ± 5%; N = 6), suggesting contributions of both Y1 and Y2 receptor activation, respectively. Y1 and Y2 receptor expression in arterial smooth muscle cells was confirmed by immunocytochemistry, and western blotting of artery lysates. α,β-meATP evoked vasoconstrictions (EC50 282 ± 32 nM; N = 6) were abolished by suramin (IC50 825 ± 45 nM; N = 5) and NF449 (IC50 24 ± 5 nM; N = 5), suggesting P2X1 mediates vasoconstriction in these arteries. P2X1, P2X4 and P2X7 were detectable by RT-PCR. Significant facilitation (1.6-fold) of α,β-meATP-evoked vasoconstrictions was observed when submaximal NPY (10 nM) was applied between α,β-meATP applications. Facilitation was antagonised by either BIBO03304 or BIIE0246. These data reveal NPY causes direct vasoconstriction in human arteries which is dependent upon both Y1 and Y2 receptor activation. NPY also acts as a modulator, facilitating P2X1-dependent vasoconstriction. Though in contrast to the direct vasoconstrictor effects of NPY, there is redundancy between Y1 and Y2 receptor activation to achieve the facilitatory effect

Contrasting phenotypes of putative proprioceptive and nociceptive trigeminal neurons innervating jaw muscle in rat

BACKGROUND: Despite the clinical significance of muscle pain, and the extensive investigation of the properties of muscle afferent fibers, there has been little study of the ion channels on sensory neurons that innervate muscle. In this study, we have fluorescently tagged sensory neurons that innervate the masseter muscle, which is unique because cell bodies for its muscle spindles are in a brainstem nucleus (mesencephalic nucleus of the 5(th )cranial nerve, MeV) while all its other sensory afferents are in the trigeminal ganglion (TG). We examine the hypothesis that certain molecules proposed to be used selectively by nociceptors fail to express on muscle spindles afferents but appear on other afferents from the same muscle. RESULTS: MeV muscle afferents perfectly fit expectations of cells with a non-nociceptive sensory modality: Opiates failed to inhibit calcium channel currents (I(Ca)) in 90% of MeV neurons, although I(Ca )were inhibited by GABA(B )receptor activation. All MeV afferents had brief (1 msec) action potentials driven solely by tetrodotoxin (TTX)-sensitive Na channels and no MeV afferent expressed either of three ion channels (TRPV1, P2X3, and ASIC3) thought to be transducers for nociceptive stimuli, although they did express other ATP and acid-sensing channels. Trigeminal masseter afferents were much more diverse. Virtually all of them expressed at least one, and often several, of the three putative nociceptive transducer channels, but the mix varied from cell to cell. Calcium currents in 80% of the neurons were measurably inhibited by μ-opioids, but the extent of inhibition varied greatly. Almost all TG masseter afferents expressed some TTX-insensitive sodium currents, but the amount compared to TTX sensitive sodium current varied, as did the duration of action potentials. CONCLUSION: Most masseter muscle afferents that are not muscle spindle afferents express molecules that are considered characteristic of nociceptors, but these putative muscle nociceptors are molecularly diverse. This heterogeneity may reflect the mixture of metabosensitive afferents which can also signal noxious stimuli and purely nociceptive afferents characteristic of muscle

Neuropeptide Y facilitates P2X1 receptor-dependent vasoconstriction via Y1 receptor activation in small mesenteric arteries during sympathetic neurogenic responses

ATP, norepinephrine and NPY are co-released by sympathetic nerves innervating arteries. ATP elicits vasoconstriction via activation of smooth muscle P2X receptors. The functional interaction between neuropeptide Y (NPY) and P2X receptors in arteries is not known. In this study we investigate the effect of NPY on P2X1-dependent vasoconstriction in mouse mesenteric arteries. Suramin or P2X1 antagonist NF449 abolished α,β-meATP evoked vasoconstrictions. NPY lacked any direct vasoconstrictor effect but facilitated the vasoconstrictive response to α,β-meATP. Mesenteric arteries expressed Y1 and Y4 receptors, but not Y2 or Y5. Y1 receptor inhibition (BIBO3304) reversed NPY facilitation of the α,β-meATP-evoked vasoconstriction. L-type Ca2+ channel antagonism (nifedipine) had no effect on α,β-meATP-evoked vasoconstrictions, but completely reversed NPY facilitation. Electrical field stimulation evoked sympathetic neurogenic vasoconstriction. Neurogenic responses were dependent upon dual α1-adrenergic (prazosin) and P2X1 (NF449) receptor activation. Y1 receptor antagonism partially reduced neurogenic vasoconstriction. Isolation of the P2X1 component by α1-adrenergic blockade allowed faciliatory effects of Y1 receptor activation to be explored. Y1 receptor antagonism reduced the P2X1 receptor component during neurogenic vasoconstriction. α1-adrenergic and P2X1 receptors are post-junctional receptors during sympathetic neurogenic vasoconstriction in mesenteric arteries. In conclusion, we have identified that NPY lacks a direct vasoconstrictor effect in mesenteric arteries but can facilitate vasoconstriction by enhancing the activity of P2X1, following activation by exogenous agonists or during sympathetic nerve stimulation. The mechanism of P2X1 facilitation by NPY involved activation of the NPY Y1 receptor and the L-type Ca2+ channel

In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization

Treating pain by inhibiting ATP activation of P2X3-containing receptors heralds an exciting new approach to pain management, and Afferent's program marks the vanguard in a new class of drugs poised to explore this approach to meet the significant unmet needs in pain management. P2X3 receptor subunits are expressed predominately and selectively in so-called C- and Aδ-fiber primary afferent neurons in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body. P2X3 antagonists block the activation of these fibers by ATP and stand to offer an alternative approach to the management of pain and discomfort. In addition, P2X3 is expressed pre-synaptically at central terminals of C-fiber afferent neurons, where ATP further sensitizes transmission of painful signals. As a result of the selectivity of the expression of P2X3, there is a lower likelihood of adverse effects in the brain, gastrointestinal, or cardiovascular tissues, effects which remain limiting factors for many existing pain therapeutics. In the periphery, ATP (the factor that triggers P2X3 receptor activation) can be released from various cells as a result of tissue inflammation, injury or stress, as well as visceral organ distension, and stimulate these local nociceptors. The P2X3 receptor rationale has aroused a formidable level of investigation producing many reports that clarify the potential role of ATP as a pain mediator, in chronic sensitized states in particular, and has piqued the interest of pharmaceutical companies. P2X receptor-mediated afferent activation has been implicated in inflammatory, visceral, and neuropathic pain states, as well as in airways hyperreactivity, migraine, itch, and cancer pain. It is well appreciated that oftentimes new mechanisms translate poorly from models into clinical efficacy and effectiveness; however, the breadth of activity seen from P2X3 inhibition in models offers a realistic chance that this novel mechanism to inhibit afferent nerve sensitization may find its place in the sun and bring some merciful relief to the torment of persistent discomfort and pain. The development philosophy at Afferent is to conduct proof of concept patient studies and best identify target patient groups that may benefit from this new intervention

The role of the purinergic P2X7 receptor in renal haemodynamic physiology and hypertensive renovascular injury

Purinergic signaling regulates numerous intrarenal mechanisms contributing to the long-term control of blood pressure. The P2X7 receptor is an ionotropic receptor activated by extra-cellular ATP that participates in inflammation and activation of immune cells. P2X7 is also expressed in the vascular endothelium, including in the kidney where its function remains poorly understood. Previous research identified the gene P2rx7, which encodes the P2X7 receptor, as a candidate gene for susceptibility to hypertensive renal vascular injury in Fischer (F344) rats. Higher expression of P2X7 was reported in several rat models of hypertensive kidney injury and both pharmacological blockade and P2X7 deletion were found renoprotective. However, studies raised concerns regarding the specificity of the broadly used P2X7 antagonists which have generated conflicting results. Here, I hypothesized that global genetic deletion of P2X7 in F344 rats will prevent the development of renal inflammation and injury in a model of chronic angiotensin II (Ang II)-induced hypertension. In this study, I sought to characterize the contribution of P2X7 to basal renal vascular and tubular functions in male and female rats. I also aimed to determine whether absence of P2X7 exerts renoprotective effects during chronic Ang II infusion, and to provide cellular and molecular mechanistic evidence for such protection using a combination of in vivo, ex vivo and in vitro studies. To this end, I used F344 rats to generate a novel CRISPR-Cas9-designed P2X7 knockout (KO) and showed that it is a true global KO with no functional P2X7 protein expressed. Using wire myography, I found that P2X7 KO impaired endothelial-dependent vasodilation in the renal artery of male, but not female, rats that may reflect diminished nitric oxide (NO) production in response to acetylcholine. NO is a key paracrine factor regulating the renal pressure natriuresis mechanism and blood pressure (BP). Thus, I assessed kidney function in anesthetized male and female P2X7 KO and wild-type (WT) rats at baseline and following acute stepwise increases in renal perfusion pressure. Overall, results showed no significant genotype effect on renal haemodynamics, BP and NO production. Moreover, my data do not support a major role of P2X7 in the modulation of tubular sodium reabsorption. I next attempted to generate an experimental model of chronic Ang II infusion-induced hypertensive kidney and vascular injury in F344 WT and P2X7 KO rats. After 5-6 weeks of infusion, rats developed modest renal damage consisting of perivascular fibrosis and tubular injury. P2X7 did not contribute to the development of renal perivascular fibrosis and tubular injury during chronic Ang II infusion. Finally, I investigated the implication of P2X7 to endothelial-to-mesenchymal transition (EndMT) using the potent P2X7 antagonist A438079 in vitro. I found that P2X7 blockade did not affect EndMT. In summary, I propose that P2X7 receptors modestly promote endothelial NO production in healthy rat renal arteries and inhibit tubular sodium reabsorption in a sex-specific manner. The role of P2X7 in renal vascular functions may become more relevant in a pathological context and requires further investigations with better disease models

ATP and mechanisms of central CO2 chemosensitivity

ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release

ATP and mechanisms of central CO2 chemosensitivity

ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Multi-Scale Peripheral Vasculopathy with Metabolic Syndrome

The combination of cardiovascular and metabolic risk factors including obesity, dyslipidemia, hypertension, and insulin resistance, in combination with a prothrombotic and proinflammatory state, is a condition termed Metabolic Syndrome (METS). Twenty percent of the adult population is afflicted with METS which increases the risk of type-2 diabetes mellitus and cardiovascular disease. Further, the presence of peripheral vascular disease (PVD) is tightly coupled with METS which is a perfusion-demand mismatch of blood supply to active skeletal muscle resulting in painful claudication and a late-stage potential for amputation. The underlying contributors of METS associated micro-vasculopathies in the skeletal muscle, their impact on impaired perfusion, and the potential for reversibility remain unclear. Owing its hyperphagia to leptin signaling resistance, the obese Zucker rat (OZR) is a translationally relevant model for human METS and the associated micro-vasculopathies. The overall purpose of this thesis is to utilize a multi-scale approach, particularly intravital microscopy and isolate vessels, to garner a greater understanding of the observed OZR vasculopathies and to investigate the potential of therapeutic interventions for their reversibility. Project 1: The purpose was to identify any alterations in postcapillary and collecting venule function in the OZR compared to healthy controls. The OZR presented with impaired dilator reactivity and elevation in thromboxane A2 constrictor responses for both postcapillary and collecting venules. Project 2: The purpose was to identify the possible contributors of a disconnect for in-situ and ex-vivo vascular studies utilizing the OZR model. Using a multi-scale approach, Project 2 provides insight to this disconnect and reveals a heterogenous adrenergic response in the OZR, giving rise to new potential avenues of study. Project 3: The purpose was to determine the potential for reversibility or restoration of established PVD using the chronic ingestion of an HMG-CoA inhibitor, atorvastatin, and/or the implementation of regular exercise. Following a seven-week intervention, the intervention groups revealed vascular improvements with the combination group having the greatest capacity for reversibility (in specific indices). Significance: Therefore, this thesis further advances the understanding of METS associated PVD as well as potential modes for improvement following its establishment

On the early onset of vascular stiffening and sexual dimorphism of sympathetic control in the spontaneously hypertensive rat

The purpose of this thesis was to explore the role of the sympathetic nervous system (SNS) in hindlimb vasomotor control during the development of hypertension (HT). Using an animal model of essential HT (the spontaneously hypertensive rat [SHR]) we demonstrated that neuropeptide Y (NPY) and the Y1 receptor (Y1R) play a greater role in modulating hindlimb hemodynamics in the early stages of HT compared to normotensive controls (Wistar Kyoto [WKY]). Hindlimb vascular mechanics (compliance [C] and viscoelasticity [K]) were assessed using a modified Windkessel model developed in our laboratory. The hindlimb mechanics did not appear to be regulated by NPY or the Y1R specifically, but the SNS did appear to regulate the hindlimb mechanics in both SHRs and WKY animals. The use of female animals in physiologic research is limited, thus the role of the SNS in developing HT in females is unknown. Finally, differences in the hemodynamic and hindlimb vascular mechanics between male and female animals were examined. Female animals exhibited augmented MAP and HR relative to males in conjunction with greater stiffness and viscoelasticity in both SHR and WKY animals. Female SHRs also appeared to lose SNS control over the stiffness and viscoelastic properties of the hindlimb vascular bed, while male SHRs maintained this control. These sexually dimorphic characteristics provide evidence for a novel proposed mechanism of cardiovascular regulation in young female rats