Notes: Abstract  The effects of the putative selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate (βγme-L-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. βγMe-L-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 μM. In contrast, the archetypal P2X purinoceptor agonist, α,β-methylene ATP (αβmeATP; 1–100 μM), produced concentration-related depolarisation responses with a mean EC50 value of 10.8 μM. The depolarising effects of αβmeATP were not attenuated by βγme-L-ATP (100 μM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 μM), but not βγme-L-ATP (1–300 μM), evoked rapid (〈20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, βγme-D-ATP and αβmeATP competed with high affinity for [3H]αβmeATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, βγme-L-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 μM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, βγme-L-ATP (1–100 μM) and αβmeATP (0.3–100 μM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 μM, respectively. βγMe-L-ATP (1–10 μM) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [3H]αβmeATP exhibited high affinity for βγme-L-ATP, αβmeATP and βγme-D-ATP with mean pIC50 values of 7.7, 8.4 and 7.3, respectively. These results indicate that βγme-L-ATP exhibits neither agonist nor antagonist properties at P2X purinoceptors on rat vagal neurones and possesses only very low affinity for [3H]αβmeATP binding sites in rat brain. In contrast, βγme-L-ATP is a potent, high affinity agonist at smooth muscle P2X purinoceptors of the rat vas deferens. This selective agonist action of βγme-L-ATP suggests that P2X purinoceptors in smooth muscle and neurones are different and represent distinct P2X purinoceptor subtypes.

Notes: Abstract The effects of the putative selective P2X purinoceptor agonist, β,γ-methylene-l-adenosine 5′-triphosphate (βγme-l-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. βγMe-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 μM. In contrast, the archetypal P2X purinoceptor agonist, α, β methylene ATP (αβmeATP;1–100 μM), produced concentration-related depolarisation responses with a mean EC50 value of 10.8 μM. The depolarising effects of αβmeATP were not attenuated by βγme-l-ATP (100 μM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 μM), but not βγme-l.-ATP (1–300 μM), evoked rapid ( 〈 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, βγme-d-ATP and αβmeATP competed with high affinity for [3H]Lx βmeATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, βγme-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 μM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, βγme-l-ATP (1–100 μM) and αβmeATP (0.3–100 μM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 μM, respectively. βγMe-l-ATP (1–10 μM) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [3H]αβmeATP exhibited high affinity for ββ γme-l-ATP, αβmeATP and βγme-d-ATP with mean PIC50 values of 7.7, 8.4 and 7.3, respectively. These results indicate that βγme-l-ATP exhibits neither agonist nor antagonist properties at P2X purinoceptors on rat vagal neurones and possesses only very low affinity for [3H]αβmeATP binding sites in rat brain. In contrast, βγme-l-ATP is a potent, high affinity agonist at smooth muscle P2X purinoceptors of the rat vas deferens. This selective agonist action of βγme-l-ATP suggests that P2X purinoceptors in smooth muscle and neurones are different and represent distinct P2X purinoceptor subtypes.

Notes: Abstract This review compares the activity of the plant transposable elements Ac, Tam3, En/Spm and Mu in heterologous plant species and in their original host. Mutational analysis of the autonomous transposable elements and two-element systems have supplied data that revealed some fundamental properties of the transposition mechanism. Functional parts of Ac and En/Spm were detected by in vitro binding studies of purified transposase protein and have been tested for their importance in the function of these transposable elements in heterologous plant species. Experiments that have been carried out to regulate the activity of the Ac transposable element are in progress and preliminary results have been compiled. Perspectives for manipulated transposable elements in transposon tagging strategies within heterologous plant species are discussed.

Notes: Abstract Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the β-glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.

Notes: Summary Radioligand binding studies have been performed to study the distribution of the binding sites for the P2x purinoceptor selective agonist radioligand, [3H]α,β-methylene ATP ([3H]αβ-meATP), in membranes prepared from various peripheral organs and several brain regions of the rat. In agreement with previous studies in the rat vas deferens, [3H]αβ-meATP labelled two populations of sites. One site exhibited high affinity for the ligand (Kd = 0.7 nM; Bmax = 1012 fmol.mg−1 protein) while the other site exhibited lower affinity (Kd = 70.8 nM) and higher capacity (Bmax = 7470 fmol. mg−1 protein). In competition studies, using a low concentration of radioligand (1 nM), the high affinity αβ-meATP binding sites in vas deferens membranes could be preferentially labelled (84–91%). Under these conditions, the PZx purinoceptor agonists, αβ-meATP and β,γ-methylene ATP, had the highest affinity with pIC50 values of 8.3 and 7.3 respectively. The P2y purinoceptor agonist, 2-methyl-thio-ATP (2-me-S-ATP), had lower affinity (pIC50 = 6.7), while uridine triphosphate, adenosine diphosphate and adenosine, agonists at the P2u, P2t and P1 purinoceptors, respectively, possessed low affinity (pIC50 values 〈 5.6). In addition, the P2 purinoceptor antagonists, cibacron blue and suramin, inhibited binding over the same concentration range at which they behave as functional antagonists at the P2x purinoceptor. High and low affinity binding sites for [3H]αβ-meATP were also identified in a range of other peripheral tissues (spleen, heart and liver) and in several brain regions (striatum, cerebral cortex, hippocampus). In the spleen, heart, cerebral cortex and liver the Kd values at both the high affinity binding sites (Kd = 1 – 1.2 nM) and the low affinity binding sites (Kd = 98 – 158 nM) were similar to the respective Kd values at the high and low affinity binding sites in the vas deferens. In competition studies performed using a low concentration of radioligand (1 nM) these sites exhibited a similar pharmacological profile to that seen in the vas deferens. Detailed analysis of competition curves to several of the ATP analogues in each of the tissues revealed that the binding profile of the radioligand was complex since several compounds, and in particular ATP and 2-meS-ATP, identified a lower proportion of sites with high affinity than did αβ-meATP The simplest interpretation of these data is that there are both high and low affinity sites for [3H]αβ-meATP in all tissues, but that the high affinity sites display heterogeneity with respect to various purinoceptor agonists. We conclude that this study demonstrates the presence of high affinity binding sites for [3H]αβ-meATP in a range of peripheral tissues and in several brain regions. These sites display a pharmacological profile which suggests that they reflect binding to a P2x purinoceptor. The apparent heterogeneity of these high affinity sites may be a consequence of agonist-induced conformational changes which we speculate may be linked with desensitisation states of the receptor.

Notes: Abstract In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [3H]α,β-methylene ATP ([3H]αβmeATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [3H]αβmeATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 μM), produced the greatest increases in total binding of [3H]αβmeATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 μM and 1–10 mM, increased total binding of [3H]αβmeATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC50 values for potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturation studies. In the absence of added divalent cations, [3H]αβmeATP labelled both high (pKd = 9.15) and low (pKd = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pKd = 9.56) and by 30 μM ZnCl2 (pKd = 9.46) but not by 3 mM MgCl2. The Bmax of the low affinity site for [3H]αβmeATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 μM ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl2 using a low concentration of [3H]αβmeATP (1 nM); the sites exhibited the binding characteristics expected of the P2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl2 and using a high concentration of [3H]αβmeATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [3H]αβmeATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, αβmeATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [3H]αβmeATP binding sites than for the low affinity binding sites. Secondly, GTP-γ-S and pyrophosphate were selective ligands for the low affinity [3H]αβmeATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC50 values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [3H]αβmeATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity. In conclusion the present study has demonstrated that cations have a marked effect on the binding of [3H]αβmeATP in rat vas deferens. Of particular interest was the ability of CaCl2 to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl2 to increase the Bmax of the low affinity binding sites enabled these sites to be selectively labelled.