Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30–60 min inhibited P2Y₁ receptor protein, only weakly modulated P2X₁, P2X₂ and P2X₃, and up‐regulated by about two‐fold P2X₄, P2X₇ and P2Y₄. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X₇ and P2Y₄ proteins, but not P2X₄ or P2Y₁. Glucose starvation transiently increased (up to three‐fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10‐fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst‐positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia‐induced expression of P2X₇ and P2Y₄. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst‐positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia‐induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors

Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y₆ antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X₇ antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y₆ and P2X₇ receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca²⁺ in T cells. The P2X₇ and P2Y₆ receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y₆ and P2X₇ receptors contributes to T cell activation via TCR.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X_1–7 and metabotropic P2Y_{1,2,4,6,11–14} receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y_11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X_5,6 and P2Y_1,6,14 in striatum), medium (P2X₃ in striatum and substantia nigra, P2X_6,7 and P2Y₁ in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y_2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X_1,3,4,6 on GABAergic neurons and P2Y₄ on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease.     

Nav 1.8-null mice show stimulus-dependent deficits in spinal neuronal activity

Extracellular ATP is known to mediate synaptic transmission as a neurotransmitter or a neuromodulator via ionotropic P2X and metabotropic P2Y receptors. Several lines of evidence have suggested that ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors, in which the P2X₃ subtype is considered as an important candidate for the effect. Conversely, we previously found that the activation of supraspinal P2X receptors evoked antinociception. However, the subtypes responsible for the antinociception via supraspinal P2X receptors remain unclear. In the present study, we showed that intracerebroventricular (i.c.v.) pretreatment with A-317491 (1 nmol), the novel non-nucleotide antagonist selective for P2X₃ and P2X_2/3 receptors, attenuated the antinociceptive effect produced by i.c.v. administered α,β-methylene-ATP (10 nmol), the P2X receptor agonist, in rats. Similarly, the abolishment of the P2X₃ receptor mRNA in the brainstem by repeated i.c.v. pretreatments with antisense oligodeoxynucleotide for P2X₃ gene once a day for 5 consecutive days diminished the antinociceptive effect of α,β-methylene-ATP. Furthermore, i.c.v. administration of A-317491 (1 and 10 nmol) significantly enhanced the inflammatory nociceptive behaviors induced by the intraplantar injection of formalin and intraperitoneal injection of acetic acid. Taken together, these results suggest that supraspinal P2X₃/P2X_2/3 receptors play an inhibitory role in pain transmission.     

Localisation of P2Y<Subscript>1</Subscript> and P2Y<Subscript>4</Subscript> receptors in dorsal root,nodose and trigeminal ganglia of the rat

The presence and distribution of P2Y (nucleotide) receptor subtypes in rat sensory neurons has been investigated. RT-PCR showed that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptor mRNA is expressed in sensory ganglia [dorsal root ganglion (DRG), nodose ganglion (NG) and trigeminal ganglion (TG)]. The regional and cellular distribution of P2Y₁ and P2Y₄ receptor proteins in these ganglia was investigated using immunohistochemistry. P2Y₁ polyclonal antibodies stained over 80% of the sensory neurons, particularly the small-diameter (neurofilament-negative) neurons. The P2Y₄ receptor antibody stained more medium- and large- (neurofilament-positive) diameter neurons than small-diameter neurons. P2Y₁ and P2Y₄ receptor immunoreactivity (P2Y₁-IR and P2Y₄-IR) was often coexpressed with P2X₃ receptor immunoreactivity (P2X₃-IR) in subpopulations of neurons. Double immunohistochemistry showed that 73–84% of P2X₃ receptor-positive neurons also stained for the P2Y₁ receptor in DRG, TG and NG while only 25–35% also stained for the P2Y₄ receptor. Subpopulations of P2Y₁-IR neurons were coexpressed with NF200, CGRP and IB₄; most P2Y₄-IR neurons were coexpressed with NF200, while only a few neurons were coexpressed with CGRP (10–20%) or with IB₄ (1–2%). The results suggest that P2Y as well as P2X receptor subtypes contribute to purinergic signalling in sensory ganglia.     

Modification of neuropathic pain sensation through microglial ATP receptors

Neuropathic pain that typically develops when peripheral nerves are damaged through surgery, bone compression in cancer, diabetes, or infection is a major factor causing impaired quality of life in millions of people worldwide. Recently, there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia. It was shown in an animal model of neuropathic pain that microglial P2X₄ and P2X₇ receptors are crucial in pain signaling after peripheral nerve lesion. In this review, we describe the modification of neuropathic pain sensation through microglial P2X₄ and P2X₇, with the possibility of P2Y₆ and P2Y₁₂ involvement.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Expression of P2X6 receptors in the enteric nervous system of the rat gastrointestinal tract

Expression of P2X₄ and P2X₆ receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X₆ receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X₆ receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X₆ receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X₆ receptors. P2X₆ receptors do not form homomultimers, but rather heteromultimers with either P2X₂ or P2X₄ receptors. P2X₄ receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X₆ receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X₆ receptors probably in heteromeric combination with P2X₂ receptors, may be involved in regulating the physiological functions of these neurons.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X₇ receptors. GSC exposure for 48 h to 10–150 μM ATP, P2R ligand, or to ADPβS or MRS2365, P2Y₁R agonists, enhanced cell expansion. This effect was counteracted by the PY₁R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y_2/4R, or 2′(3′)-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X₇R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A₃R or P2X₇R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A₃R, P2Y₁R, and P2X₇R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.     

Expression of P2Y receptors in the rat anterior pituitary

In this study, the distribution patterns of P2Y₁, P2Y₂ P2Y₄, P2Y₆, P2Y₁₂, and P2Y₁₃ receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y₁ and P2Y₄ receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y₂ receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y₆ receptors were expressed in macrophages. P2Y₁₃ receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y₁ and P2Y₄ receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y₁ and P2Y₄ receptors, some lactotrophs via P2Y₂ receptors, and folliculo-stellate cells via P2Y₂ receptors in the rat anterior pituitary.