Stable electric-field driven cone-jetting of concentrated biosuspensions.

Electrospraying, or electrohydrodynamic jetting, is one of several jet-based technologies being explored to process living biological organisms. One of the key advantages of electrospraying is its ability to deposit advanced materials with high resolution that cannot be obtained with other competing technologies, such as ink-jet printing. However, to generate a controlled droplet size distribution in the micrometre range necessary for precision drop and placement of materials requires jetting in stable cone-jet mode. In this paper, we describe the experimental set-up and conditions by which electrospray jetting in stable cone-jet is achieved and use this methodology to process a highly concentrated biological suspension having 10(7) cells ml(-1), the highest cellular loading processed to this day by a jetting approach in this jet based category. The areas of study to which this technology may be applied span the physical and the life sciences.

Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes.

Transcripts for P2X(2) and P2X(6) subunits are present in rat CNS and frequently colocalize in the same brainstem nuclei. When rat P2X(2) (rP2X(2)) and rat P2X(6) (rP2X(6)) receptors were expressed individually in Xenopus oocytes and studied under voltage-clamp conditions, only homomeric rP2X(2) receptors were fully functional and gave rise to large inward currents (2-3 microA) to extracellular ATP. Coexpression of rP2X(2) and rP2X(6) subunits in Xenopus oocytes resulted in a heteromeric rP2X(2/6) receptor, which showed a significantly different phenotype from the wild-type rP2X(2) receptor. Differences included reduction in agonist potencies and, in some cases (e.g., Ap(4)A), significant loss of agonist activity. ATP-evoked inward currents were biphasic at the heteromeric rP2X(2/6) receptor, particularly when Zn(2+) ions were present or extracellular pH was lowered. The pH range was narrower for H(+) enhancement of ATP responses at the heteromeric rP2X(2/6) receptor. Also, H(+) ions inhibited ATP responses at low pH levels (

Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors.

In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.

Expression of P2 receptors in bone and cultured bone cells.

Extracellular nucleotides acting through P2 receptors elicit a wide range of responses in many cell types. There is increasing evidence that adenosine triphosphate (ATP) may function as an important local messenger in bone and cartilage. In this study, we used immunocytochemistry, employing novel polyclonal antibodies against P2X(1-7) receptors, and in situ hybridization, using oligonucleotide probes corresponding to P2X(2,4) and P2Y(2,4) messenger RNAs (mRNAs), to localize P2 receptors on undecalcified bone sections and on cultured osteoblasts and osteoclasts. We provide the first direct evidence that the P2X(2) receptor subtype is expressed on osteoclasts, osteoblasts, and chondrocytes. We also obtained evidence for the expression of P2X(5) and P2Y(2) receptors on osteoblasts and chondrocytes, and for P2X(4) and P2X(7) receptors on osteoclasts. Our results confirm earlier reports of P2Y(2) and P2X(4) expression in human osteoclastoma and rabbit osteoclasts, respectively, and are consistent with ATP responses observed on bone cells using electrophysiological techniques. Our novel finding that P2X(2) is expressed by osteoclasts is of particular interest. P2X(2) is the only P2 receptor subtype that requires extracellular acidification to show its full sensitivity to ATP, and our recent functional studies have shown that the stimulatory action of ATP on resorption pit formation by mature osteoclasts is amplified greatly at low pH. These findings point to fundamental new mechanisms for the local modulation of bone resorption.

Synergy between the inositol phosphate responses to transfected human adenosine A1-receptors and constitutive P2-purinoceptors in CHO-K1 cells.

1. The effect of adenosine A1-receptor and P2-purinoceptor agonists on [3H]-inositol phosphate accumulation has been investigated in CHO-K1 cells transfected with the human adenosine A1-receptor. 2. Adenosine receptor agonists stimulated [3H]-inositol phosphate accumulation in CHO-K1 cells with a rank potency order of N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > N6-2-(4-aminophenyl) ethyladenosine (APNEA). The responses to both CPA and APNEA were antagonized by the A1 selective antagonist, 1,3-dipropylcyclopentylxanthine (DPCPX) yielding KD values of 1.2 nM and 4.3 nM respectively. 3. ATP, UTP and ATP gamma S were also able to stimulate [3H]-inositol phosphate accumulation in these cells with EC50 values of 1.9 microM, 1.3 microM and 5.0 microM respectively. 2-Methyl-thio-ATP was a weak agonist of this response (EC50 > 100 microM). 4. The [3H]-inositol phosphate response to CPA was completely attenuated by pertussis toxin treatment (24 h; 100 ng ml-1). In contrast, the responses to ATP, UTP and ATP gamma S were only reduced by circa 30% in pertussis toxin-treated cells. 5. The simultaneous addition of CPA and either ATP, UTP or ATP gamma S produced a large augmentation of [3H]-inositol phospholipid hydrolysis. This was due to an increase in the maximal response and was significantly greater than the predicted additive response for activation of these two receptor systems. The synergy was not observed in pertussis toxin-treated cells. 6. No synergy was observed between the [3H]-inositol phosphate responses to histamine and ATP in CHO-K1 cells transfected with the bovine histamine H1-receptor. In these cells the response to histamine was completely resistant to inhibition by pertussis toxin treatment. 7. This study provides a clear demonstration of a synergy between pertussis toxin-sensitive and insensitive receptor systems in a model cell system which is an ideal host for transfected cDNA sequences. This model system should provide a unique opportunity to unravel the mechanisms underlying this example of receptor cross-talk involving phospholipase C.

Thermodynamics of full agonist, partial agonist, and antagonist binding to wild-type and mutant adenosine A1 receptors.

A thermodynamic analysis of the binding of a full agonist (N6-cyclopentyladenosine), a partial agonist (8-butylamino-N6-cyclopentyladenosine) and an antagonist (8-cyclopentyltheophylline) to human wild-type and mutant (mutation of a threonine (Thr) to an alanine (Ala) residue at position 277) adenosine A1 receptors expressed on Chinese hamster ovary (CHO) cells, and to rat brain adenosine A1 receptors was undertaken. The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium to rat brain receptors were determined by means of affinity measurements carried out at four different temperatures (0, 10, 20 and 25 degrees) and van't Hoff plots. Two temperatures (0 and 25 degrees) were considered for human receptors. Affinity constants were obtained from inhibition assays on membrane preparations of rat brain and CHO cells by use of the antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as selective adenosine A1 receptor radioligand. As for rat brain receptors, full agonist binding was totally entropy driven, whereas antagonist binding was essentially enthalpy driven. Partial agonist binding appeared both enthalpy and entropy driven. As for human receptors, full agonist affinity was highly dependent on the presence of Thr277. Moreover, affinity to both wild-type and mutant receptors was enhanced by temperature increase, suggesting a totally entropy-driven binding. Antagonist binding did not depend on the presence of Thr277. Antagonist affinity decreased with an increase in temperature, suggesting a mainly enthalpy-driven binding. Partial agonist binding was significantly dependent on the presence of Thr277 at 25 degrees, whereas such a dependence was not evident at 0 degrees. It is concluded that Thr277 contributes only to the binding of adenosine derivatives and that its role changes drastically with the receptor conformation and with the type of agonist (full or partial) interacting with the adenosine A1 receptors.

Molecular cloning and characterisation of a human brain A1 adenosine receptor cDNA.

Using the sequence conservation in the G protein-coupled receptor superfamily, we have isolated an adenosine A1 receptor cDNA from a human hippocampal cDNA library by homology screening. When expressed in mammalian CHO.K1 cells, the protein encoded by this cDNA binds the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) with high affinity (Kd = 0.56 +/- 0.11 nM) and, functionally, is able to inhibit cAMP production upon receptor activation with the A1-specific agonist N6-cyclopentyladenosine (CPA) (> 80% inhibition at 10(-7) M CPA). The binding and functional characteristics of the expressed cDNA demonstrate that we have isolated a human brain adenosine receptor cDNA of the A1 subtype.

Novel G protein-coupled receptors: a gene family of putative human olfactory receptor sequences.

We have taken advantage of the sequence conservation in the G protein-coupled receptor superfamily to isolate a fragment of a novel G protein-coupled receptor sequence using polymerase chain reaction (PCR) amplification of human genomic DNA. Screening of human genomic and hippocampal cDNA libraries with this amplified receptor fragment revealed a number of related sequences. Sequence analysis of four genomic clones and one cDNA clone clearly identifies these as related members of the G protein-coupled receptor family, as the deduced amino acid sequence reveals putative transmembrane domains and conserved amino acid residues. Southern blot analysis of restriction digests of human genomic DNA indicates that these receptor subtypes are likely to belong to a family of related genes. One of the proposed receptor sequences indicates the presence of pseudogenes in this family. Based on the homology of these sequences to a family of recently described receptors expressed exclusively in rat olfactory epithelium, it is suggested that these receptors represent a family of human odorant receptors.

Molecular cloning, functional characterization and possible cooperativity between the murine P2X4 and P2X4a receptors.

We have cloned and functionally characterised the mouse orthologue of the P2X4 receptor, mP2X4, and a splice variant of this receptor, mP2X4a. mP2X4 is 388 amino acids in length and shares 94% and 87% identity with the rat and human P2X4 receptors, respectively, while mP2X4a is 361 amino acids in length and lacks a 27-amino acid region in the extracellular domain corresponding to exon 6 of the known P2X receptor gene structures. When expressed in Xenopus laevis oocytes, mP2X4 produces a rapid inward current in response to ATP with an EC50 of 1.68+/-0.2 microM, consistent with the affinity of the rat and human P2X4 receptors for ATP. This agonist response is potentiated by the P2X receptor antagonists suramin, Reactive blue 2 and, over a limited concentration range, by PPADS. Although mP2X4a forms a poorly functional homomeric receptor, it appears able to interact with the full-length mP2X4 subunit to result in a functional channel with a reduced affinity for ATP. These results suggest a possible role for splice variants of P2X receptors in the formation of functional heteromeric ion channels.

Cloning, characterisation and chromosomal assignment of the human adenosine A3 receptor (ADORA3) gene.

The gene for the inhibitory G-protein coupled human A3 adenosine receptor (ADORA3) was isolated and sequence analysis shows that the coding region is interrupted by a single intron of size 2.4 kb. The location of this intron in the second intracellular loop is conserved with respect to the A1, A2a and A2b adenosine receptor subtype genes. The ADORA3 gene was mapped to 1p13.3 by fluorescence in situ hybridisation. Northern blot studies show that the gene is widely expressed and is most abundant in brain and some endocrine tissues. We have mapped multiple transcription start sites in two cell lines and lung tissue by primer extension and 5' RACE (rapid amplification of cDNA ends). The ADORA3 gene promoter lacks CAAT and TATA boxes but has putative binding sites for multiple transcription factors. In contrast to the A1 adenosine receptor gene we find no evidence of alternate splicing in the 5' untranslated region of the ADORA3 gene.

A threonine residue in the seventh transmembrane domain of the human A1 adenosine receptor mediates specific agonist binding.

The A1 adenosine receptor is a member of the seven-transmembrane G protein-coupled, receptor superfamily. This receptor binds the purine nucleoside adenosine with high affinity and inhibits the activity of adenylate cyclase. We have used site-directed mutagenesis and functional expression studies to examine the role of the threonine residue, located at position 277 in transmembrane domain VII of the human A1 receptor. Mutation of Thr-277 to either serine or alanine resulted in the expression of receptors that had essentially no change in binding affinity for the A1 selective antagonist 8-cyclo-pentyl-1,3-dipropylxanthine. Mutation of Thr-277 to serine resulted in modest (4.4-8.6-fold) but significant increases in the observed Ki values for three adenosine agonists, namely N-(1-methyl-2-phenethyl)adenosine (R-PIA or S-PIA) and 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-L- ribofuranuronamide) (NECA). However, mutation of Thr-277 to alanine resulted in no significant changes in the Ki for R-PIA or S-PIA but did result in a highly significant 437-fold increase in the Ki for NECA. This demonstrates that the hydroxyl moiety of Thr-277 mediates agonist but not antagonist binding and, more specifically, that this residue forms a probable molecular contact site with the 5' substitution found in NECA.

Recombinant P2Y receptors: the UCL experience.

The beginning of the last decade heralded three important and sequential developments in our understanding of cell-to-cell signalling by extracellular ATP via its cell surface receptors, the P2 purinoceptors. One major development in ATP signalling culminated in a timely review in 1991, when it was established in the clearest of terms that ATP receptors exploited discrete signal transduction pathways (Dubyak, G.R., 1991. Signal transduction by P2-purinergic receptors for extracellular ATP. Am. J. Respir. Cell. Mol. Biol. 4, 295-300; and later in Dubyak, G.R., El-Moatassim, C., 1993. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577-C606). Henceforth, it was universally acknowledged that some P2 purinoceptors interacted with heterotrimeric G-proteins to activate intracellular signalling cascades (metabotropic ATP receptors), whereas others contained intrinsic ion-channels (ionotropic ATP receptors). A second key development can be traced to 1992, from the discovery that ATP receptors were involved in excitatory neurotransmission in the CNS and PNS (Edwards, F.A., Gibb, A.J., Colquhoun, D., 1992. ATP receptor-mediated synaptic currents in the central nervous system. Nature 359, 144-147; Evans, R.J., Derkach, V., Surprenant, A., 1992. ATP mediates fast synaptic transmission in mammalian neurons. Nature 357, 503-505; Silinsky, E.M., Gerzanich, V., Vanner, S.M., 1992. ATP mediates excitatory synaptic transmission in mammalian neurones. Br. J. Pharmacol., 106, 762-763). Thereafter, it was accepted that ATP could play a neurotransmitter and/or modulatory role throughout the entire nervous system. The third key development stemmed from the isolation of a cDNA, from chick brain, encoding a metabotropic ATP receptor (Webb, T.E., Simon, J., Krishek, B.J., Bateson, A.N., Smart, T.G., King, B.F., Burnstock, G., Barnard, E.A., 1993. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 324, 219-225). The cloning of a membrane protein serving as an ATP receptor ignited a widespread international interest in purinergic signalling. Investigators at University College London (UCL) - colleagues and associates of Geoffrey Burnstock - were at the forefront of this rapid phase of discovery. In this review, we highlight the UCL experience when the fields of molecular biology, physiology and cell biology converged to help advance our understanding of ATP as an extracellular signalling molecule.

P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study.

The expression of the seven P2X receptor subtypes and of two P2Y receptors was examined immunohistochemically and by in situ hybridisation in thymi of adult male rats. P2X4, P2Y2 and 4 receptor mRNA colocalisation studies combining in situ hybridisation and immunohistochemistry were also carried out. P2X and P2Y receptors were found on thymocytes. P2X receptors were also abundant in cells of the thymic microenvironment, involved in control of T-cell maturation in vivo. We are the first to describe the expression of P2X4 receptors on thymocytes and confirm the finding of P2X1 and P2Y2 receptors on subpopulations of lymphocytes. P2X1,2,3,4 and 5 receptors were present in blood vessels of the thymus. P2X1,2 and 4 receptors were detected in vascular smooth muscle, while P2X3 receptors appeared to be associated with endothelial cells; some small arteries were positive for P2X5, possibly labelling vascular smooth muscle or fibroblasts in the adventitia. P2X2,3,6 and 7 receptors were found on thymic epithelial cells. P2X2 and 3 receptors were abundant on medullary epithelial cells, whilst P2X6 receptors were prominent in Hassall's corpuscles. P2X2 receptors were found on subcapsular and perivascular epithelial cells. P2X2,6 and 7 receptors were detected in epithelial cells along the thymic septa. Expression of P2X receptors was also investigated by Western blotting of crude thymic tissue extracts under reducing conditions. All seven P2X receptor subtypes were found to be dimers of approximately 70 kDa and 140 kDa molecular weight. ATP-mediated apoptosis and cell proliferation of thymocytes are discussed.

Selective expression of purinoceptor cP2Y1 suggests a role for nucleotide signalling in development of the chick embryo.

Responses to extracellular nucleotides (e.g., ATP, ADP, etc.) have been demonstrated in a number of embryonic cell types suggesting they may be important signalling molecules during embryonic development. Here the authors describe for the first time the expression of a G-protein-coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1), during embryonic development of the chick. During the first 10 days of embryonic development, cP2Y1 is expressed in a developmentally regulated manner in the limb buds, mesonephros, brain, somites, and facial primordia, suggesting that this receptor may have a role in the development of each of these systems.

Antagonism of an adenosine/ATP receptor in follicular Xenopus oocytes.

Follicular Xenopus oocytes possess a novel receptor where both adenosine and ATP activate a cAMP-dependent, nonrectifying K+-current. Five compounds, alpha,beta-methylene ATP (alpha, beta-meATP), 8-(p-sulfophenyl)theophylline (8-SPT), theophylline, 2, 2'-pyridylisatogen tosylate (PIT) and suramin, were tested as antagonists of adenosine- and ATP-activated K+-currents. The descending order of activity (pIC50 values) against adenosine responses was: alpha,beta-meATP (6.72) = 8-SPT (6.68) > theophylline (5.32) > PIT (4.58), whereas suramin was relatively inactive. The blocking actions of alpha,beta-meATP and alkylxanthine compounds were reversible with washout, whereas blockade by PIT was irreversible. These antagonists showed similar blocking activity against ATP responses, except for PIT which was more effective at ATP responses than at adenosine responses. The selectivity of antagonists was tested against cAMP-dependent K+-currents evoked by forskolin and follicle-stimulating hormone (FSH). 8-SPT and theophylline did not inhibit but instead augmented forskolin and FSH responses; this augmentation may be caused by inhibition of phosphodiesterase activity inside follicle cells. On the other hand, alpha,beta-MeATP and PIT inhibited forskolin and FSH responses; both compounds apparently are nonselective antagonists. Thus, only alkylxanthine derivatives (8-SPT and theophylline) were selective antagonists of the novel adenosine/ATP receptor in Xenopus oocytes, whereas alpha,beta-meATP and PIT were nonselective in their blocking actions and suramin was relatively inactive.

Characterization and chromosomal localization of the human A2a adenosine receptor gene: ADORA2A.

The gene for the stimulatory G protein-coupled human A2a adenosine receptor was isolated and sequence analysis revealed two exons that are interrupted by an intron of approximately 6.4 kb. An intron is located in the same region in the human A1 and A2b adenosine receptor genes. Comparison of the A2a genomic and cDNA sequences reveals two nucleotide differences in the coding region and the presence of an aberrant sequence in the 5'208 base pairs of the A2a cDNA including a polymorphism in the third base of codon Tyr-361 and Gly codon which was always detected at residue 392, indicated that the Arg codon present in the cDNA may be an artifact. Fluorescent in situ hybridization and PCR analysis of human-hamster hybrid cell panels shows that the A2a receptor gene is localized to chromosome 22q11.2. This is in contrast with previous reports (subsequently retracted) which mapped the A2a gene to chromosome 11q11-13.

Coexpression of P2X(3) and P2X(2) receptor subunits in varying amounts generates heterogeneous populations of P2X receptors that evoke a spectrum of agonist responses comparable to that seen in sensory neurons.

Using voltage-clamp procedures on Xenopus oocytes, agonist-evoked ionic currents by P2X receptors resulting from the coexpression of P2X(2) and P2X(3) subunits were compared against the agonist responses of homomeric P2X(2) and P2X(3) receptors. With the quantity of P2X(3) mRNA kept constant and quantity of P2X(2) mRNA progressively increased, expressed P2X receptors changed from a P2X(3)-like receptor to a P2X(2)-like receptor. In all cases, however, agonist-evoked responses comprised biphasic (fast and slow) currents-the former showing the properties of P2X(3) receptors and latter consistent with the presence of P2X(2) and P2X(2/3) receptors. Using desensitization procedures, the P2X(3)-like fast current was selectively removed to allow the slow current to be studied in isolation. P2X(2/3) receptors were then characterized by slowly inactivating inward currents that were reproducible within 30 s of washout and whose pharmacological profile [selective agonists, Ap(5)A > alpha,beta-methylene ATP >> beta,gamma-methylene ATP > UTP; antagonists, TNP-ATP >> suramin > or = Reactive blue-2 (RB-2)] contrasted with the profile of P2X(2) receptors (Ap(5)A, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and UTP inactive; antagonists, RB-2 > TNP-ATP > suramin). Thus, our experiments reveal that coexpression of two P2X subunits, which of themselves can generate functional homomeric receptors, results in a complex population of heterogeneous P2X receptors-in this case P2X(2), P2X(3), and P2X(2/3) receptors. Depending on the relative levels of P2X subunit coexpression, the operational profile of the resultant P2X receptors can change from one phenotype to another. This spectrum may explain the variability of agonist responses in small sensory neurons that also express P2X(2) and P2X(3) subunits in different amounts.

A functional screening of adenosine analogues at the adenosine A2B receptor: a search for potent agonists.

Various adenosine analogues were tested at the adenosine A2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2B receptors. 5'-N-Substituted carboxamidoadenosines were most potent. 5'-N-Ethylcarboxamidoadenosine (NECA) was most active with an EC50 value of 3.1 microM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8- and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3- and 7-deazaanalogues were not active.

Diimidazo[1,2-c:4',5'-e]pyrimidines: adenosine agonist activity demonstrated by microphysiometry.

Silicon-based microphysiometry, measuring extracellular acidification rate of cells in culture, demonstrated that a series of diimidazo[1,2-c:4',5'-e]pyrimidines were agonists at the human adenosine A1 receptor. 5-amino-7,8-dihydro-3-ribofuranose-8-(R)-(phenyl)-3H-diimidazo [1,2-c:4',5'-e]pyrimidine (2a) had an EC50 of 100 microM and reached 90% of the Emax produced by R-PIA.