Two‐component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two‐component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock‐out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene‐regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium‐regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

1 Previous studies have shown that the rat duodenum relaxes to adenosine and adenosine 5'-triphosphate (ATP) via P1 and P2Y purinoceptors respectively, but in preliminary studies uridine 5'-triphosphate (UTP) was found to contract this tissue. The non-selective P2 antagonist suramin and a number of nucleotides were therefore used to investigate this response further.2 ATP, UTP, adenosine 5'-diphosphate (ADP), adenosine 5'-O-(3-thiotriphosphate) (ATP-7-S), guanosine 5'-triphosphate (GTP) and uridine 5'-diphosphate (UDP) each relaxed the duodenum, with an agonist potency order of ATP = ADP>ATP-7-S>>GTP>>UTP = UDP, consistent with the presence of a P2Y purinoceptor mediating relaxation. 3 ATP-i-S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP-y-S > UTP> UDP, although maximal responses to these agonists were not obtained at a concentration of 267 JAM (ATP-i-S) and 300 pM (UTP and UDP). No contractions were observed with any of the other agonists at concentrations up to 300 JM. 4 Indomethacin (25 JM) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins. 5 Suramin (100 JAM and mM) inhibited relaxations induced by ATP, shifting the concentration-response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1 mM). Suramin (1 mM) inhibited relaxations induced by ATP-j-S, shifting the concentration-response curve to the right, and completely abolished contractions induced by ATP-y-S. In contrast, suramin (100 JAM and 1 mM) had no effect on contractions induced by UTP. Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1IJM). In the presence of suramin (I mM), no contractions to ATP were observed.6 These results confirm that in the rat duodenum there is a P2Y purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP-y-S act to cause contraction. Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin-insensitive pyrimidinoceptor as well as a suramin-sensitive receptor for ATP-y-S. An alternative explanation is that the differential effects of suramin are via its actions as an antagonist in addition to its action as an ectonucleotidase inhibitor.

1 Previous studies have shown that the rat duodenum contains PI and P2y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-y-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 4 Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-y-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-y-S, and no cross-desensitization between AMPCPP and UTP or ATP-y-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramininsensitive.5 ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-Me-SAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-y-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-y-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6 These results show that there is a P2y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-y-S, and at which ATP and UTP show equal potency, and is thus probably of the P2u subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this degradation is unlikely to explain the differential antagonism by suramin of the nucleotides.

1 Adenosine 5'-triphosphate (ATP) and adenosine have been shown to contract the rat colon muscularis mucosae, and the receptors at which they act have been classified as P2y and Al respectively.Uridine 5'-triphosphate (UTP) also contracts this tissue, and desensitization was used to investigate the receptors by which it acts, in the light of recent suggestions that specific pyrimidinoceptors may exist for UTP, or that nucleotide receptors may exist which are responsive to both ATP and UTP but not to some ATP analogues such as 2-methylthioadenosine 5'-triphosphate (2-MeSATP). 2 ATP, UTP and adenosine each contracted the rat colon muscularis mucosae in a concentrationdependent manner over the concentration range 0.3-300 ltM, although maximal responses to ATP and UTP were not obtained. ATP was approximately 4 times as potent as UTP and approximately equipotent with adenosine although the maximal response to adenosine appeared to be less than that to ATP or UTP.3 Desensitization of the tissue with ATP (200 gM) given immediately before each concentration of the agonists reduced subsequent contractions induced by ATP itself and also by UTP, but did not reduce contractions induced by adenosine. Desensitization of the tissues with UTP (20011M) also reduced contractions induced by ATP and UTP but not by adenosine, whereas desensitization with adenosine (200 iM) reduced contractions induced by adenosine itself but not by ATP or UTP. 4 Desensitization of the tissue with 2-MeSATP (200 iLM), which is a more potent agonist than ATP at P2Y-purinoceptors, greatly reduced the responses to ATP and to UTP, but had no effect on responses induced by adenosine. Attempts to desensitize the tissue with adenosine 5'-(a,P-methylene)triphosphonate (AMPCPP), which is a more potent agonist than ATP at P2X-purinoceptors but is less potent at P2y-purinoceptors, were unsuccessful. 5 These results show that cross desensitization to ATP and UTP occurred and was specific for these agonists rather than being due to a general decrease in the ability of the muscle to contract. This implies that ATP and UTP act at the same receptor, which does not support the existence of specific pyrimidinoceptors but which could be taken as evidence for the existence of a nucleotide receptor on this tissue. However, the ability of 2-MeSATP, which is inactive at the proposed nucleotide receptors, also selectively to desensitize this receptor indicates instead that ATP and UTP are both acting at a purinoceptor of the P2Y type in this tissue.

The functional effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), adenosine 5'-tetraphosphate (AP4) and the diadenosine polyphosphates P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A) and P1,P5-diadenosine pentaphosphate (Ap5A) were studied in two isolated smooth muscle preparations thought to contain P2Y (P2Y1) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap3A=Ap4A> ATP>AP4=Ap5A, and these relaxations were antagonised by suramin with apparent pA2 values in the region of 5, consistent with activation of a P2Y1 receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap3A = Ap4A>ATP=AP4 =Ap5A >UTP. However, while suramin (100 microM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP4, Ap3A and Ap4A and had little effect on contractions induced by Ap5A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap3A, Ap4A, Ap5A and AP4. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP4, Ap3A, Ap4A and Ap5A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A1 receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin.