The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.

P2x-and P2y-purinoceptors in the guinea-pig urinary bladder and taenia coli respectively. Potentiation of purinoceptor-mediated activity was seen only at a low concentration of suramin (1 yM) and only in the urinary bladder (P2x-purinoceptor). For its antagonistic activity suramin did not discriminate between P2X-and P2y-purinoceptors, but it was selective for P2-purinoceptor-mediated activity rather than that mediated via cholinoceptors, adrenoceptors or histamine receptors.

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.

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1 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an inhibitor of P2x-purinoceptormediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by a,B-methylene ATP (a,P-MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle.2 PPADS (1-30 gM) caused concentration-dependent inhibition of contractions to the stable P2X-purinoceptor agonist, cx,,-MeATP, decreasing the maximum response to a,4-MeATP (30 JAM) at concentrations of 3-30 JiM. The pD2 value for a,4-MeATP in the absence of PPADS was 6.52 ± 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 gM the negative log concentrations of a,4-MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 ± 0.09 (8), 5.64 ± 0.12 (7), 5.15 ± 0.23 (7) and 4.78 ± 0.22 (5). 3 PPADS (1-30IM) caused concentration-dependent inhibition of contractions to stimulation of intramural purinergic nerves (1 -32 Hz). There was a greater inhibition at lower frequencies (1-8 Hz)than at higher frequencies (16-32 Hz). PPADS, 30 JAM, did not produce significantly greater antagonism than 1OIJM. 4 PPADS (30 jAM) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 ± 0.16 (5) and 6.33 ± 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 6 Thus, PPADS (1-30 JM) antagonized responses mediated via P2X-purinoceptors in the rabbit urinary bladder. It was selective for P2-purinoceptor-mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2x-purinoceptors.

1. The isolated perfused rat mesenteric arterial bed was used to examine the activity of the adenine dinucleotides: beta‐nicotinamide adenine dinucleotide (NAD); beta‐nicotinamide adenine dinucleotide phosphate (NADP); flavin adenine dinucleotide (FAD); and of the alpha,omega‐diadenosine polyphosphates: adenylyl adenosine (AP1A); P1,P2‐diadenosine pyrophosphate (AP2A); P1,P3‐diadenosine triphosphate (AP3A); P1,P4‐diadenosine tetraphosphate (AP4A); P1,P5‐diadenosine pentaphosphate (AP5A); P1,P6‐diadenosine hexaphosphate (AP6A). Responses were compared with those of ADP, ATP, 2‐methylthio‐ATP (2‐meSATP) and alpha,beta‐methylene ATP (alpha,beta‐meATP). 2. In basal tone preparations mono‐ and dinucleotides elicited vasoconstriction with the order of potency: alpha,beta‐meATP > or = AP5A > or = AP6A > or = AP4A > or = 2‐meSATP >> ATP >> ADP. The dinucleotides NAD, NADP, FAD, AP1A, AP2A and AP3A had no effect. 3. The P2X‐purinoceptor antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulphonic acid (30 microM) virtually abolished vasoconstrictor responses to AP4A, AP5A and AP6A. 4. Auto‐ and cross‐desensitization of vasoconstrictor responses to AP4A, AP5A, AP6A, ATP and alpha,beta‐meATP were observed. 5. In raised tone preparations nucleotides elicited endothelium‐dependent vasodilatation with the order of potency: 2‐meSATP = ADP > ATP > AP3A > AP2A > AP1A = NADP = FAD > NAD. The nucleotides AP4A, AP5A, AP6A and alpha,beta‐meATP had no vasodilator effects. 6. It is concluded that the alpha,omega‐adenine dinucleotides AP4A, AP5A and AP6A elicit vasoconstriction, but not vasodilatation, in the rat mesenteric arterial bed via P2x‐purinoceptors. In contrast, the dinucleotides NADP, FAD, AP1A, AP2A and AP3A elicit vasodilatation, but not vasoconstriction, via endothelial P2Y‐purinoceptors. 7. It is suggested that there is a crucial relationship between the structure of the alpha,omega‐diadenosine polyphosphates and their activity at P2X‐ and P2Y‐purinoceptors with a pivotal role played by the polyphosphate chain. Molecules with four or more phosphates are vasoconstrictors, while those with three or less phosphates are vasodilators.