Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of p2 proteins

The replication proteins encoded in the P2 region of the poliovirus genome induce extensive rearrangement of cellular membranes into vesicles and are a required component of viral RNA replication complexes. To identify distinct viral protein(s) from the P2 region of the genome that were required to form functional RNA replication complexes, the P2 proteins were expressed in addition to P3 in HeLa S10 translation-RNA replication reactions. Membrane-associated preinitiation replication complexes were isolated from these reactions and used to measure negative-strand synthesis. The formation of replication complexes capable of initiating negative-strand synthesis was observed when either P23 or when P2 and P3 were expressed in the HeLa S10 translation-replication reactions. The amount of negative-strand RNA synthesized with P2 and P3 was approximately 50% of that observed with P23. Negative-strand synthesis was not observed when the processed forms of the P2 proteins (e.g., 2A, 2B, 2C, 2AB, and 2BC) were used in various combinations in place of P2. In contrast, the expression of 2A and 2BCP3 supported negative-strand synthesis at the same level observed with P23. Therefore, functional replication complexes were formed in reaction mixtures that contained either 2A and 2BCP3 or P2 and P3. Genetic complementation analysis of P23 RNA that contained a lethal mutation in 2C confirmed these results. The expression of 2BCP3 in trans restored the replication of P23-2C(P131N) RNA to wild-type levels. The expression of P2 and P3 also complemented the replication of this mutant RNA, although very inefficiently. Complementation was not observed in reactions that contained P2 alone, 2BC, or 2C. Based on these results, we propose that RNA replication complexes are initially formed with the primary cleavage products of P23 (i.e., P2 and P3 or 2A and 2BCP3), and that 2A and 2BCP3 are preferentially used in this process.     

Distribution of P2X receptors in the rat adrenal gland

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.     

P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity

The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1β, P2α and P2β, which form two heterodimers, P1α/P2β and P1β/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1β-P2β heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1β/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2β-P0-P1β/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.     

Functional Properties of Five Dictyostelium discoideum P2X Receptors

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Structural properties of the human acidic ribosomal P proteins forming the P1-P2 heterocomplex

The ribosome has a morphologically distinct structural feature called the stalk, recognized as a vital element for its function. The ribosomal P proteins constitute the main part of the eukaryotic ribosomal stalk, forming a pentameric structure P0-(P1-P2)(2). The group of P1/P2 proteins in eukaryotes is very diverse, and in spite of functional and structural similarities they do not fully complement one another, probably constituting an adaptive feature of the ribosome from a particular species to diverse environmental conditions. The functional differences among the P1/P2 proteins were analysed in vivo several times; however, a thorough molecular characterization was only done for the yeast P1/P2 proteins. Here, we report a biophysical analysis of the human P1 and P2 proteins, applying mass spectrometry, CD and fluorescence spectroscopy, cross-linking and size exclusion chromatography. The human P1/P2 proteins form stable heterodimer, as it is the case for P1/P2 from yeast. However, unlike the yeast complex P1A-P2B, the human P1-P2 dimer showed a three-state transition mechanism, suggesting that an intermediate species may exist in solution.     

Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.     

Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase carrying ribulose 1,5-bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose-1,5-bisphosphate-carboxylase/oxygenase activase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate-binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate-binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246-252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30 degrees C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50 degrees C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0-8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO. The ATP-hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3-5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.     

Human sperm protamines. Amino-acid sequences of two forms of protamine P2

Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium X HCl containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gas-phase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine P1. The amino acid sequence of P2a is: (sequence; see text)     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Stable overexpression of specific segments of the P2P-R protein in human MCF-7 cells promotes camptothecin-induced apoptosis

The stable overexpression of near full-length P2P-R protein in human Saos 2 cells restricts cell cycle progression by inducing mitotic arrest at prometaphase and mitotic apoptosis (Gao and Scott, 2002). Those effects of P2P-R were observed in Saos-2 cells that lack p53 and employ a caspase-3-dependent apoptotic signaling pathway. The current studies were performed to evaluate if overexpression of specific segments of the P2P-R protein promote apoptosis in human MCF-7 cells that contain p53 and employ a different apoptotic signaling pathway. Since segments of P2P-R were found not to induce apoptosis independently, the ability of three different P2P-R segments to promote camptothecin-induced apoptosis was evaluated following their stable transfection and expression in MCF-7 cells. Relative to full-length P2P-R (1-1560 aa), the three P2P-R segments used in these studies included: P2P-R-2 (761-1560 aa), P2P-R-3 (1156-1560 aa), and P2P-R-4 (1314-1560 aa). The results document that overexpression of P2P-R-2 and P2P-R-3 promotes camptothecin-induced apoptosis by three to fivefold when assayed by flow cytometric analysis of apoptotic sub 2n cell populations or by TUNEL assays. In contrast, P2P-R-4 had no effect on apoptosis. These results suggest that the ability of P2P-R to promote camptothecin-induced apoptosis in MCF-7 cells involves a specific region (1156-1314 aa) that exists within P2P-R. The data presented also show that the p53 binding domain of P2P-R overlaps with the apoptosis-associated region and previous studies documented that this region of P2P-R also binds single-strand nucleotides (Witte and Scott, 1997). Therefore, P2P-R-promoted apoptosis induced by camptothecin may be influenced by such interactions.     

P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation

Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 > P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.     

Mapping of attenuating sequences of an avirulent poliovirus type 2 strain.

A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.     

Phosphorylation and N-terminal region of yeast ribosomal protein P1 mediate its degradation, which is prevented by protein P2

The stalk proteins P1 and P2, which are fundamental for ribosome activity, are the only ribosomal components for which there is a cytoplasmic pool. Accumulation of these two proteins is differentially regulated in Saccharomyces cerevisiae by degradation. In the absence of P2, the amount of P1 is drastically reduced; in contrast, P2 proteins are not affected by a deficiency in P1. However, association with P2 protects P1 proteins. The half-life of P1 is a few minutes, while that of P2 is several hours. The proteasome is not involved in the degradation of P1 proteins. The different sensitivity to degradation of these two proteins is associated with two structural features: phosphorylation and N-terminus structure. A phosphorylation site at the C-terminus is required for P1 proteolysis. P2 proteins, despite being phosphorylated, are protected by their N-terminal peptide. An exchange of the first five amino acids between the two types of protein makes P1 resistant and P2 sensitive to degradation.     

Comparative Analysis of P2X1, P2X2, P2X3, and P2X4 Receptor Subunits in Rat Nodose Ganglion Neurons

Nodose ganglion (NG) neurons are visceral primary sensory neurons. The transmission and regulation of visceral sensation is mediated mainly by the P2X purinoceptor (P2X receptor). Although the characteristics of different P2X receptor subunits in the NG have been studied previously, comprehensive analyses have not been performed. In this study, we used immunohistochemistry, immunocytochemistry, and whole cell patch clamp techniques to compare the expression and function of P2X1, P2X2, P2X3, and P2X4 receptor subunits in adult rat NG neurons. Polyclonal antibodies against the four P2X subunits labeled different subpopulations of NG neurons. P2X1 and P2X3 were expressed mainly in small-to-medium sized NG neurons, whereas P2X2 and P2X4 were located mostly in medium- and larger-sized NG neurons. Over 36% of NG neurons were P2X3 positive, which was higher than the other three P2X subunits. In addition, different types of currents were recorded from neurons expressing different P2X subunits. The fast type of ATP current was recorded from neurons containing P2X1–4 subunits, the intermediate type of current was recorded from neurons containing the P2X1, P2X3, and P2X4 subunits, the slow type was recorded from neurons expressing P2X1–3, and/or P2X4 subunits, whereas the very slow type was recorded from neurons containing the P2X2 and P2X3 subunits. These comparative results provide an anatomical verification of the different subunits in NG neurons, and offer direct support for the idea that various functional NG populations have distinct responses to ATP, which might be in part due to the different expression profiles of diverse P2X subunits.     

Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure.

We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor mRNA-expression was quantified in hearts from CHF rats and compared to sham-operated rats. Furthermore, the presence of receptor mRNA was studied in the myocardium from patients with heart failure. In the sham operated rats the G-protein coupled P2Y-receptors were expressed at a higher level than the ligand gated ion-channel receptor (P2X1). Among the P2Y-receptors the P2Y6-receptor was most abundantly expressed (P2Y6 > P2Y1 > P2Y2 = P2Y4 > P2X1). A prominent change was seen for the P2X1- and P2Y2-receptor mRNA levels which were increased 2.7-fold and 4.7-fold respectively in the myocardium from the left ventricle of CHF-rats. In contrast, the P2Y1-, P2Y4- and P2Y6-receptor mRNA levels were not significantly altered in CHF rats. In human myocard the P2X1-, P2Y1-, P2Y2-, P2Y6- and P2Y11-receptors were detected by RT-PCR in both right and left atria and ventricles, while the P2Y4-receptor band was weak or absent. In conclusion, most of the studied P2-receptors were expressed in both rat and human hearts. Furthermore, the P2X1- and P2Y2-receptor mRNA were upregulated in CHF, suggesting a pathophysiological role for these receptors in the development of heart failure.