Role Of Purinoceptors Research Articles

Introduction to the Special Issue on Purinergic Receptors.

In this Introduction to the series of papers that follow about purinergic receptors, there is a brief history of the discovery of purinergic signalling, the identity of purinoceptors and the current recognition of P1, P2X and P2Y subtypes. An account of key functions mediated by purinoceptors follows, including examples of both short-term and long-term (trophic) signalling and a table showing the selective agonists and antagonists for the purinoceptor subtypes. References to evolution and roles of purinoceptors in pathological conditions are also presented.

P2X4 Receptors of Microglia in Neuropathic Pain

We have learned various data on the role of purinoceptors (P2X4, P2X7, P2Y6 and P2Y12 receptors) expressed in spinal microglia and several factors that presumably activate microglia in neuropathic pain after peripheral nerve injury. Especially P2X4 receptors (P2X4Rs) make a critical contribution to the pain processing. P2X4Rs of microglia might be promising targets for treating neuropathic pain. A predicted therapeutic benefit of interfering with microglial P2X4Rs may be that normal pain sensitivity would be unaffected since expression or activity of most of these receptors are upregulated or enhanced predominantly in activated microglia in the spinal cord where damaged sensory fibers project. Recently, we found that CCL21 regulates the expression of P2X4Rs in different manners, respectively. These new findings also provide novel targets for developing anti-neuropathic pain medicines.

Role of Purinoceptors in Regulating Afferent Arteriolar Diameter in DOCA‐salt Hypertensive Rats

Afferent arterioles (AA) are highly responsive to purinoceptor stimulation. ATP contributes importantly to renal autoregulation by activating P2 receptors. Pressure‐mediated, and ATP and β,γ‐methylene ATP (P2X1 receptor agonist)‐induced AA vasoconstriction is impaired in DOCA‐salt hypertensive rats (DOCA rats) while the function of other purinoceptors is unclear. We postulated that P2Y and A1 receptor reactivity is intact in AA from DOCA rats. AA responses to UTP (a P2Y2 receptor agonist) and adenosine were studied in juxtamedullary nephrons. Systolic blood pressure averaged 125±5 and 180±7 mmHg in uninephrectomized (UNx) and DOCA rats, respectively. UTP (10−8 to 10−4 mol/L, n=5) evoked concentration‐dependent vasoconstriction of AA in UNx rats. Diameter declined by 2±1, 6±2, 12±2, 23±5 and 49±9%, respectively. UTP‐induced vasoconstriction was unchanged in DOCA rats (n=5, p>0.05) leading to declines of 3±2, 10±2, 14±3, 29±5 and 43±9%, respectively. The AA response to adenosine was also indistinguishable between two groups (n=6 each, p>0.05). Preglomerular microvascular P2X1 receptor protein expression was unaltered in DOCA rats. These data demonstrate that AA P2Y2 and A1 receptor signaling is preserved in DOCA rats. This study suggests that impairment of AA autoregulatory behavior in DOCA‐salt hypertension cannot be attributed to impaired P2Y2 and A1 receptor signaling.

Involvement of the purinergic system in central cardiovascular modulation at the level of the nucleus ambiguus of anaesthetized rats

Anatomical studies have demonstrated the existence of purinergic P2 receptors in the nucleus ambiguus (NA), a site containing cardiac vagal motoneurons. However, very little is known about the functional role of these receptors in central cardiac vagal regulation. The aims of our study were to evaluate the following: (1) the blood pressure and heart rate responses following purinoceptor activation within the NA; (2) the role of purinoceptors and excitatory amino acid (EAA) receptors in mediating the cardiovascular responses evoked by ATP and L-glutamate stimulation of NA; and (3) the role of NA purinoceptors in mediating the cardiovascular responses of the Bezold-Jarisch reflex. In anaesthetized rats, microinjection of L-glutamate (5.0 nmol/50 nl) into the NA induced a marked and immediate onset bradycardia with minimal change in arterial pressure. Microinjection of ATP into the NA induced a dose-dependent (0.31-6.0 nmol/50 nl) bradycardia and pressor responses. It is noteworthy that the bradycardia occurred either before or simultaneously with a pressor response (when present), indicating that it was not a baroreceptor reflex mediated response due to the rise in arterial pressure. The pressor response was prevented by α(1)-adrenergic blockade with prazosin, whereas muscarinic blockade with methyl-atropine abolished the evoked bradycardia. Ipsilateral microinjection of PPADS (a P2 receptor antagonist; 500 pmol/100 nl) into the NA significantly attenuated the ATP-induced bradycardia but spared the pressor response. In contrast, PPADS in the NA had no effect on the L-glutamate-evoked bradycardic response. Ipsilateral injection of kynurenic acid (a non-selective EAA receptor antagonist; 10 nmol/50 nl) into the NA totally blocked the bradycardia induced by l-glutamate and partly attenuated the ATP induced bradycardia. Finally, both the depressor and the bradycardic responses of the Bezold-Jarisch reflex were attenuated significantly (P < 0.01 and P < 0.05, respectively) following bilateral microinjection of PPADS into the NA. These results identify ATP and purinergic P2 receptors within the ventrolateral medulla as excitatory to cardiovagal neurons. Additionally, our data show that P2 receptors within the ventrolateral medulla are integral to the cardiovascular responses of the Bezold-Jarisch reflex.

Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT

Microglia are implicated as a source of diverse proinflammatory factors in the CNS. Extracellular nucleotides are well known to be potent activators of glial cells and trigger the release of cytokines from microglia through purinergic receptors. However, little is known about the role of purinoceptors in microglial chemokine release. In this study, we found that high concentrations of ATP evoked release of CC-chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1alpha from MG-5 cells, a mouse microglial cell line, and rapid up-regulation of CCL3 mRNA was elicited within 30 min of ATP stimulation. The release of CCL3 was also stimulated by 2'- and 3'-O-(4-benzoylbenzoyl) ATP, an agonist of P2X(7) receptors. Brilliant Blue G, an antagonist of P2X(7) receptors, strongly inhibited this ATP-induced CCL3 release. Similar pharmacological profile was observed in primary microglia. In MG-5 cells, ATP caused de-phosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). ATP-induced NFAT de-phosphorylation was also dependent on P2X(7) receptor activation. Furthermore, ATP-induced CCL3 release and production were prevented by a selective inhibitor of NFAT. Taken together, the results of this study demonstrate an involvement of NFAT in the mechanism underlying P2X(7) receptor-mediated CCL3 release.

Purinoceptors in the Kidney

The multiple roles of extracellular ATP and its metabolite adenosine include broad areas, such as regulating vascular tone and inducing inflammation. This review will discuss purinoceptor-induced effects on renal vascular resistance, highlighting the key experiments providing a significant contribution to our current understanding of autoregulatory mechanisms. Emphasis will be placed on the purinoceptor subtypes involved in autoregulatory control by ATP and adenosine. Additionally, the role of purinoceptors in hypertension-associated impairment of autoregulatory efficiency will be discussed.

Role of Purinoceptors in Cardiac Function in Rats during Ontogeny

Intravenous injection of exogenous ATP (10(-4) M) to rats aging 21, 56, and 100 days increased the heart rate by the 15th sec postinjection. Stable ATP analogue alpha,beta-methylene-ATP in an equimolar concentration increased the heart rate in rats aging 56 and 100 days (by the 15th second after treatment), but had no effect on 21-day-old animals. alpha,beta-Methylene-ATP in a concentration of 10(-7) M increased the heart rate in 21-day-old rat pups, which attests to high sensitivity of P2 purinoceptors. Administration of ATP and alpha,beta-methylene-ATP after treatment with nonselective purinoceptor antagonist suramin did not increase the heart rate in rats of different age groups. Infusion of ATP and its stable analogue after administration of selective P2Y receptor antagonist reactive blue 2 increased the heart rate in rats of different age groups. These changes reflect activation of P2X receptors in the heart.

The role of purinoceptors in wound healing

Purinoceptors are membrane‐bound receptors for adenosine, purines and pyrimidines that are expressed in nearly all cell types throughout the organism. Previous studies demonstrated that they are involved in the regulation of proliferation of most target cells. Since it is well known that several purinoceptors are expressed in skin keratinocytes, we were interested in examining their involvement in wound healing. Primary skin keratinocytes from four different mouse strains were isolated and cultured for in vitro studies. The expression patterns of metabotropic purinoceptors of the primary cells as well as a murine cell line were determined by RT‐PCR analysis. The specificity of the amplification products was verified by sequence analysis. Proliferation assays with various purinoceptor agonists and antagonists were performed using the murine cell line MSC‐P5. Cell proliferation was determined by incorporation of 5‐bromo‐2‐deoxyuridine (BrdU). Female NMRI mice were used for in vivo trials. To inhibit the wound healing process, animals were treated once daily with dexamethasone. After a week of treatment, full thickness wounds were set with biopsy punches in depilated back skin. The wound healing process was measured by determination of the wound area and the incorporation of BrdU in vivo. The purinoceptor agonists and antagonists were administered topically on the wound area. Among the examined metabotropic purinoceptors, the murine cell line MSC‐P5 expressed the receptors A2b, P2Y1, P2Y2 and P2Y6. Primary cultured murine keratinocytes possessed the same expression profile and they additionally expressed the P2Y4 receptor. Furthermore, three of the four primary cultures showed a splicing variant of the P2Y2 receptor. The purinoceptor agonists ATP, UTP and 5’‐(N‐ethyl)‐carboxamidoadenosine (NECA) enhanced the cell growth of the murine cell line MSC‐P5. The mitogenic effect of ATP and UTP was inhibited by the P2Y1, P2Y2 and P2Y6 receptor antagonist suramin, while the effect of NECA was inhibited by the selective A2b receptor antagonist enprofylline. Taking into account that other tested purinoceptor agonists, 2‐Me‐S‐ATP and UDP, exhibited no proliferative activity on MSC‐P5 cells, it can be concluded that ATP and UTP act on proliferation via the P2Y2 receptor, and NECA via the A2b receptor. In vivo trials in an animal model of impaired wound healing confirm that pharmacological actions via purinoceptors offer an intriguing possibility in the treatment of wounds. Nevertheless, further investigations are needed to fully elucidate the role of purinergic mechanisms involved in wound healing. Funding: Self‐funded.

Effect of diadenosine tetraphosphate (AP4A) on coronary arterial microvessels in the beating canine heart.

Diadenosine tetraphosphate (AP4A) can be released from activated platelets and the present study examined its effect on coronary arterial microvessels. The role of purinoceptors in the coronary microcirculation in vivo was also investigated. In open chest dogs, coronary arterioles were observed using a microscope with a floating objective. In Protocol 1, AP4A (1, 10, 100 and 1,000 micromol/L) was superfused onto the heart surface before and during the superfusion of 10 micromol/L of 8-phenyltheophylline (8-PT), a P1 purinoceptor blocker. In Protocol 2, AP4A (0.1, 1, 10, and 100 nmol x kg(-1) x min(-1)) was infused into the left anterior descending coronary artery before and during the superfusion of 10 micromol/L of 8-PT. In addition to 8-PT, 30 micromol/L of pyridoxalphosphate-6-azophenyl 2',4'-disulphonic acid (PPADS), a P2X purinoceptor blocker in Protocol 3, or 300 micromol/L of N(omega)-nitro-L-arginine (LNNA) in Protocol 4, was continuously superfused, and 4 doses of AP4A were cumulatively superfused as in Protocol 1. In Protocol 5, 10 micromol/L of alpha,beta-methylene ATP, an agonist of P2X purinoceptors, was superfused for 60 min. Superfused AP4A dilated arterioles in a dose-dependent manner. The magnitude of dilatation was greater in smaller arterioles (small vessel < or = 150 microm: 24.5+/-2.2% vs large vessel > 150 microm: 10.6+/-1.5% at a dose of 1,000 micromol/L, p<0.001). On the other hand, intraluminally applied AP4A also dilated arterioles, but no size dependency was shown. In the presence of 8-PT, vasodilatory responses to superfused and intraluminally applied AP4A were attenuated and the lower doses of AP4A constricted arterioles. This vasoconstrictor effect was not affected by PPADS. The vasodilatory effect of the higher doses of AP4A was almost abolished in the presence of LNNA. Alpha,beta-methylene ATP had no effect on coronary microvascular diameters. AP4A has bidirectional effects on coronary arterial microvessels: vasodilatory effects mediated by P1 purinoceptors and NO, which might be mediated by P2Y purinoceptors, and a vasoconstrictor effect, which is not mediated by P2X purinoceptors.

Purinergic responses in the feline pulmonary vascular bed

AbstractAdenosine and ATP produce dose‐dependent, tone‐dependent response in the pulmonary vascular (PV) bed, under conditions of controlled pulmonary blood flow and constant left atrial pressure in vivo in intact‐chest, spontaneously breathing cats. At low, resting PV tone, adenosine produces dose‐dependent vasoconstrictor responses by acting on “A1‐like” adenosine receptors which activate a phospholipase and thromboxane release. ATP Produces dose‐dependent vasoconstrictor responses, in part, following its metabolism to adenosine, by acting on “A1‐like” adenosine receptors and by acting on P2x receptors. At elevated PV tone, data to date support that adenosine and ATP produce dose‐dependent vasodilator responses by acting on A2 and P2Y receptors, respectively. Mechanisms to explain how a change in vascular tone alters the response of the vascular bed to a substance are not known. Moreover, the role of purinoceptors in pulmonary vascular disease is not known. Identification of selective pharmacological probes for purinoceptors in the pulmonary vascular bed is necessary to investigate their role in the development of pulmonary hypertension of acute and chronic lung disease. © 1993 Wiley‐Liss, Inc.