NUDT15 R139C-related thiopurine leukocytopenia is mediated by 6-thioguanine nucleotide-independent mechanism in Japanese patients with inflammatory bowel disease.

BACKGROUND: NUDT15 R139C (rs116855232) is a recently identified genetic factor responsible for thiopurine-induced leukocytopenia and hair loss. In this study, we investigated the association of NUDT15 R139C with 6-thioguanine nucleotide (6-TGN) levels and thiopurine-induced leukocytopenia in Japanese patients with inflammatory bowel disease (IBD). METHODS: Two hundred and sixty-four subjects (103 healthy volunteers and 161 IBD patients treated with thiopurines) were enrolled. Genotyping for NUDT15 R139C was performed using Custom TaqMan® SNP genotyping assays. RESULTS: The NUDT15 C/C, C/T, and T/T genotypes were 80.7, 18.2, and 1.1 %, respectively. The allelic frequency was 10.2 %. Among 161 IBD patients, there was no significant difference in 6-TGN levels among the NUDT15 genotypes. Forty-five patients (27.9 %) developed leukocytopenia (WBC <3000/µl), and the C/T and T/T genotypes were significantly associated with the development of leukocytopenia (P = 1.7 × 10(-5)). In these patients, 6-TGN levels were not significantly different between NUDT15 genotypes. NUDT15 R139C was significantly associated with early (<8 weeks) (P = 1.03 × 10(-4)) and late (>8 weeks) leukocytopenia (P = 4.3 × 10(-4)). The decrease in WBC count at 2 and 4 weeks was significantly higher in patients with the C/T or T/T genotypes as compared to the patients with the C/C genotype. All patients with the T/T genotype (n = 2) developed early severe hair loss and severe leukocytopenia (<1000/µl). The logistic regression analysis revealed that NUDT15 R139C was the sole genetic factor responsible for the thiopurine-induced leukocytopenia (P = 0.001). CONCLUSIONS: These results suggest that NUDT15 R139C-related thiopurine-induced leukocytopenia is mediated by a 6-TGN-independent mechanism.

Sagittal plane center of mass movement strategy and joint kinetics during sit-to-walk in elderly fallers.

BACKGROUND: Biomechanical studies have shown that the horizontal center of mass momentum at seat-off during sit-to-walk is reduced among elderly adults. However, the underlying mechanism of this phenomenon is still lacking. The purpose of this study was to examine differences in center of mass movement strategies and lower extremity joint kinetics in the elderly during sit-to-walk. METHODS: Fifteen healthy young adults, fifteen healthy elderly adults (70 years or older) and fifteen elderly fallers performed Timed Up and Go test. Biomechanical data collected from sit-to-walk phase of Timed Up and Go test were analyzed. Outcome measures included center of mass-ankle inclination angles, hip, knee and ankle joint moments, and ground reaction forces of the stance limb. FINDINGS: Results reported here are from 10 participants in each group due to missing force place data. Elderly fallers adopted a movement strategy that included a posterior foot placement at seat-off, a longer duration, and a shorter initial step length. When compared to healthy individuals, elderly fallers demonstrated a greater ankle plantarflexor moment at seat-off. INTERPRETATION: Increased ankle plantarflexor moment in elderly fallers could be a movement strategy to improve stability during sit-to-walk and may be related to the reduced ankle dorsiflexor strength commonly reported in elderly fallers. Results of this study enhance our understanding on the underlying mechanisms of the altered sit-to-walk movement and could aid in developing effective screening and rehabilitation programs to prevent falling in the elderly.

Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms.

Cephalic elevations in arterial pressure associated with microgravity and prolonged bed rest alter cerebrovascular autoregulation in humans. Using the head-down tail-suspended (HDT) rat to chronically induce headward fluid shifts and elevate cerebral artery pressure, previous work has likewise shown cerebral perfusion to be diminished. The purpose of this study was to test the hypothesis that 2 wk of HDT reduces cerebral artery vasodilation. To test this hypothesis, dose-response relations for endothelium-dependent (2-methylthioadenosine triphosphate and bradykinin) and endothelium-independent (nitroprusside) vasodilation were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10(-5) M) and cyclooxygenase inhibitor indomethacin (10(-5) M). MCA caveolin-1 protein content was measured by immunoblot analysis. Endothelium-dependent vasodilation to 2-methylthioadenosine triphosphate and bradykinin were both lower in MCAs from HDT rats. These lower vasodilator responses were abolished with N(G)-nitro-L-arginine methyl ester but were unaffected by indomethacin. In addition, HDT was associated with lower levels of MCA caveolin-1 protein. Endothelium-independent vasodilation was not altered by HDT. These results indicate that chronic cephalic fluid shifts diminish endothelium-dependent vasodilation through alterations in the endothelial nitric oxide synthase signaling mechanism. Such decrements in endothelium-dependent vasodilation of cerebral arteries could contribute to the elevations in cerebral vascular resistance and reductions in cerebral perfusion that occur after conditions of simulated microgravity in HDT rats.

Effects of forskolin on Kupffer cell production of interleukin-10 and tumor necrosis factor alpha differ from those of endogenous adenylyl cyclase activators: possible role for adenylyl cyclase 9.

Proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-alpha expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and beta-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 microg/ml) caused attenuated TNF-alpha levels in culture medium (forskolin/isoproterenol, P < or = 0.05; prostaglandin E2, P < or = 0.01). Forskolin also reduced IL-10 mRNA and protein (P < or = 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P < or = 0.05). We conclude that regulation of TNF-alpha and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2 differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9.

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor.

1. The present study was aimed at examining P2 receptor-mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 microM noradrenaline. 2. Endothelial denudation abolished the dilator responses. 3. The selective P2Y(1) agonist, 2-MeSADP, induced a potent vasodilatation (pEC(50)=6.9+/-0.1). The P2Y(1) antagonist of 10 microM, MRS 2216, shifted the 2-MeSADP concentration-response curve 1.1 log units to the right. The combined P2Y(1) and P2X agonist, 2-MeSATP, stimulated a dilatation with a potency similar to that of 2-MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2-MeSATP and 2-MeSADP indicating that P2X receptors do not mediate vasodilatation. 4. Both the P2Y(2/4) agonist, UTPgammaS and the P2Y(6) agonist, UDPbetaS, stimulated potent dilatations (pEC(50)=7.8+/-0.4 for UTPgammaS and 8.4+/-0.2 for UDPbetaS). 5. The 2-MeSADP-induced nitric oxide (NO)-mediated dilatation was studied in the presence of 10 micro M indomethacin, 50 nM charybdotoxin and 1 microM apamin. The involvement of the endothelium-derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L-NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L-NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2-MeSADP dilatation with similar efficacy (E(max)=25+/-5% for NO, 25+/-6% for EDHF and 27+/-5% for prostaglandins). 6. In conclusion, extracellular nucleotides induce endothelium-derived vasodilatation in human vessels by stimulating P2Y(1), P2Y(2/4) and P2Y(6) receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins.

Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro.

Dopaminergic mechanisms in the subthalamic nucleus (STN) are implicated in the pathophysiology of Parkinson's disease. Here, electrophysiological responses of STN neurons to dopamine (DA) were investigated by using whole-cell patch-clamp recordings in the rat brain slice preparation. Under current-clamp, DA depolarized membrane potential and increased the frequency of spontaneous action potentials of STN neurons. Under voltage-clamp, DA (3-300 microM) produced a reversible concentration-dependent inward current (I(DA); 6-40 pA) with an EC(50) of 13 microM. This DA-induced current had a negative slope conductance which reversed at -102 mV. It was partially reduced by barium and by superfusion with an elevated concentration of extracellular K(+). Moreover, TTX and glutamate receptor antagonists (CNQX and AP5) did not significantly affect the DA responses, indicating that I(DA) is not dependent upon afferent synaptic activity in the STN. Quinpirole, a D(2) receptor agonist, mimicked the DA action more effectively than did the D(1) agonist SKF-38393. The D(2) antagonist sulpiride, but not the D(1) antagonist SCH-23390, blocked responses induced by DA. Intracellular application of G-protein inhibitor GDP-beta-S also suppressed I(DA). GTP-gamma-S, added to the pipette solution, evoked a sustained inward shift in the absence of DA. These results suggest that DA increases the activity of STN neurons via activation of G-protein-coupled D(2)-like receptors which reduce a K(+) conductance.

The induction of carbon monoxide-mediated airway relaxation by PACAP 38 in isolated guinea pig airways.

Pituitary adenylate cyclase--activating peptide 38 (PACAP 38) displays several biologic activities relevant to obstructive airway disease. Carbon monoxide (CO) has recently emerged as a potent, endogenously produced mediator of bronchodilation. In this study, we have analyzed the occurrence of PACAP 38 and the corresponding occurrence of heme oxygenase (HO), the rate-limiting enzyme for CO production, in guinea pig trachea, using immunocytochemistry. We have also investigated whether the dilatory effects of PACAP 38 are dependent on CO, using an in vitro setup for tracheal studies. A moderate supply of PACAP-like immunoreactive nerve fibers was seen in association with tracheal smooth muscle. HO-like immunoreactivity was observed in the respiratory epithelium and in association with smooth muscle bundles. PACAP 38 induced a concentration-dependent relaxation of precontracted tracheal segments. This dilation was nearly abolished after pretreatment with zincprotoporphyrine, an inhibitor of heme oxygenase. The same effect was accomplished with Rp-8Br-cyclicGMPS, an inhibitor of cyclicGMP, whereas the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine had no effect on the PACAP 38--induced dilation. The presented data suggest that PACAP 38 can induce bronchodilation by means of a CO-dependent, cyclicGMP-related mechanism, thereby providing a link between neurotransmission and local CO release in the airway smooth muscle.

NO/cGMP pathway activation and membrane potential depolarization in pig ciliary epithelium.

PURPOSE: To investigate whether in isolated porcine ciliary processes, stimulation of the nitric oxide (NO)-guanylate cyclase (GC)-3',5'-cyclic guanosine monophosphate (cGMP) pathway modulates ciliary epithelial transmembrane potential. METHODS: Changes in transmembrane potential induced by the two NO donors, sodium nitroprusside (SNP; 100 microM) and S-nitroso-N-acetyl-penicillamine (SNAP; 100 microM), or by the cGMP-analogue 8-para-chlorophenylthioguanosine-3', 5'-cyclic guanosine monophosphate (8-pCPT-cGMP; 100 microM) were measured with microelectrodes in the presence or in the absence of the GC-inhibitor 1-H-(1,2,4)oxadiazole(4,3-alpha)quinoxalin-1-1 (ODQ; 10 microM). The effect of 8-pCPT-cGMP was also assessed in the presence of the anion channel inhibitors niflumic acid (100 microM), diisothiocyanatostilbene-2,2' disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), or the K+ channel blocker tetraethylammonium chloride (TEA; 10 mM). cGMP production was measured by immunoassay. RESULTS: Significant membrane depolarizations (P < 0.05-0.001; n = 5-8) were induced by SNP (6 +/- 1 mV; mean +/- SEM), SNAP (8 +/- 1 mV), or 8-pCPT-cGMP (13 +/- 1 mV). In presence of ODQ, the effect of SNP and SNAP were significantly inhibited (-2 +/- 0 mV and 0 +/- 0 mV, respectively; P < 0.05; n = 5-6), but not depolarizations elicited by 8-pCPT-cGMP. These were prevented (P < 0.05-0.01; n = 5) by niflumic acid (1 +/- 1 mV), DIDS (1 +/- 1 mV), or 9-AC (5 +/- 1 mV), but not by TEA (12 +/- 2 mV). The increase in cGMP production induced by SNP (9.5-fold) was inhibited by ODQ (P < 0.001; n = 6). CONCLUSIONS: Activation of the NO-GC-cGMP pathway modulates epithelial transmembrane potential in isolated porcine ciliary processes.

Prostaglandin E2 modulates a non-inactivating potassium current in rat neurohypophyseal nerve terminals.

A non-inactivating voltage dependent K+ channel current was observed in neuro-hypophyseal nerve terminals. This current was sensitive to inhibition by 4-aminopyridine and tetraethyl ammonium chloride, but was not sensitive to inhibition by alpha- or beta-dendrotoxin. Prostaglandin E2 (PGE2) modulated the voltage-dependent K+ channel, through a receptor-mediated process, as indicated by meclofenamate sensitivity, and this involved the activation of G protein(s), as indicated by sensitivity to guanosine-5'-O-(2-thiodiphosphate) (GDPfS). After short periods of incubation (e.g. 5 min), PGE2 increased the non-inactivating current. Following longer incubation periods with PGE2 (e.g. 20 min), the non-inactivating current declined. Forskolin and the cyclic adenosine monophosphate (AMP) analogs 8-bromo- and dibutyryl cyclic AMP, and Sp-cyclic AMPs inhibited the current, but did not mimic the increase in current caused by PGE2. Also, the cyclic AMP antagonist Rp-cyclic AMPs did not block the increase in current induced by PGE2. These results indicate that activation of cyclic AMP-dependent protein kinase (PKA) is not involved in mediating the stimulatory actions of PGE2. These observations provide evidence that PGE2 may contribute to the regulation of hormone release from the posterior pituitary by modulating K+ channels. However, the post-receptor mechanisms of subcellular signal transduction underlying this effect remain unknown.

Desensitization of the P2-purinoceptors on the rat colon muscularis mucosae.

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 A1 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 concentration-dependent manner over the concentration range 0.3-300 microM, 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 microM) 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 (200 microM) also reduced contractions induced by ATP and UTP but not by adenosine, whereas desensitization with adenosine (200 microM) reduced contractions induced by adenosine itself but not by ATP or UTP. 4. Desensitization of the tissue with 2-MeSATP (200 microM), 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'-(alpha,beta-methylene)triphosphonate(AMPCPP), which is a more potent agonist than ATP at P2X-purinoceptors but is less potent atP2y-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 specificpyrimidinoceptors 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.

Small-conductance Cl- channels in rabbit parietal cells activated by prostaglandin E2 and inhibited by GTP gamma S.

1. Small-conductance, sub-picosiemens (sub-pS) Cl- channels in the basolateral membrane of non-stimulated parietal cells in isolated rabbit gastric glands were studied by whole-cell patch-clamp and noise analysis techniques. 2. Voltage-independent whole-cell Cl- currents were recorded from parietal cells equilibrated with Cl(-)-containing solutions. Intracellular application of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, 5-200 microM) slowly decreased the whole-cell Cl- current, and its steady-state effect was observed about 6 min after the start of the dialysis. The half-maximal inhibitory concentration of GTP gamma S was about 60 microM. 3. The single Cl- channel conductance was estimated to be 0.37 pS from the variance noise analysis during the GTP gamma S-induced inhibitory process of the whole-cell Cl- current. It is in agreement with the value obtained by a method of power spectrum analysis (0.47 pS). 4. The whole-cell Cl- current was increased by prostaglandin E2 (10 microM). The increased Cl- current was reduced by the subsequent application of GTP gamma S (50 microM), whereas the GTP gamma S (50 microM)-induced inhibition of the Cl- current was not reversed by the subsequent application of prostaglandin E2 (10 microM). 5. The combined intracellular application of GTP gamma S (50 microM) and GDP beta S (500 microM) markedly reduced the inhibitory effect of GTP gamma S, indicating that a GTP-binding protein is involved in the regulation of the Cl- channel. 6. Treatment of parietal cells with pertussis toxin (PTX, 500 ng/ml) for 90-140 min did not affect the GTP gamma S-induced inhibition of the whole-cell Cl- current. 7. Intracellular application of cyclic AMP-dependent protein kinase inhibitor peptide (100 micrograms/ml) or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (5 mM, pCa 8) did not affect the GTP gamma S-induced inhibition of the whole-cell Cl- current. 8. The present study has suggested that the opening of the sub-pS Cl- channel is modulated negatively by a PTX-insensitive GTP-binding protein and positively by prostaglandin E2.

Guanylnucleotide specificity for muscarinic receptor inhibitory coupling to cardiac adenylate cyclase.

The guanylnucleotide specificity of muscarinic acetylcholine receptor (MR) inhibitory coupling to cardiac adenylate cyclase (AC) was investigated under low MgCl2 (i.e., 0.5 mM) conditions. In purified cardiac sarcolemma, carbachol maximally inhibited AC activity 60% in the presence of GTP. Carbachol-dependent inhibition in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) or guanylylimidodiphosphate [Gpp(NH)p] was of lesser magnitude (i.e., 30%) and was evident only during short incubation periods. Of greater interest, carbachol maximally inhibited AC activity in the presence of GDP and guanosine 5'-O-(2-thiodiphosphate (GDP beta S) by 35 and 60%, respectively. Control studies ruled out transphosphorylation of GDP and GDP beta S by nucleoside diphosphate kinase or guanylnucleoside triphosphate contamination as reasons for the inhibitory effects of GDP and GDP beta S. Furthermore, isoproterenol stimulated AC in the presence of GTP, GTP gamma S, and Gpp(NH)p but not in the presence of GDP or GDP beta S. Therefore, GDP and GDP beta S may serve as agonists on MR-activated Gi but not on beta-adrenergic receptor-activated Gs in these membranes. Time course studies revealed that carbachol-dependent inhibition of AC in the presence of either GTP or GDP occurred without a detectable lag period, and this inhibition was rapidly reversed by atropine. In contrast, a 1-2-min lag time was required for carbachol- and GDP beta S-dependent inhibition of AC to occur, and inhibition, once developed, was only partially and slowly reversed by atropine. Preincubation of sarcolemma with carbachol and GDP beta S, in the absence of ATP or under nonphosphorylating conditions, eliminated the lag time for inhibition of AC activity. Although it is unlikely that GDP and GDP beta S have physiological relevance of MR-Gi-AC coupling, these studies provide unique insights into this coupling mechanism in cardiac membranes.

Excitation-contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S.

1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization-induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component') produced in 'loaded' fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM- to 1 mM-GTP gamma S (guanosine-5'-O-(3-thiotriphosphate), a non-hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization-induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation-contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM-EGTA). 4. GDP beta S (guanosine-5'-O-(2-thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization-induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline-gap voltage clamp was not affected by the application of 1 mM-GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP-binding proteins (G-proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G-proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded.

Permeabilization and calcium-dependent activation of rabbit polymorphonuclear leukocytes by poly-L-arginine.

In the absence of extracellular Ca2+, poly-L-arginine induces little lysozyme release from rabbit polymorphonuclear leukocytes (PMNs). The polycation causes plasma membrane damage, which is evident from the release of the cytoplasmic enzyme lactate dehydrogenase (LDH). In the presence of Ca2+ concentrations higher than 0.2 mM, poly-L-arginine induces a strong lysozyme release that is superimposed on the membrane-damaging effect. The results suggest that poly-L-arginine permeabilizes the plasma membrane, enabling Ca2+ to enter the cell, which results in the exocytotic release of granule constituents. The GTP analog GTP gamma S shifts the Ca2+ requirement of exocytosis to slightly higher concentrations, whereas it completely inhibits poly-L-arginine-induced LDH release. Pertussis toxin gives a moderate inhibition, and La3+ completely inhibits poly-L-arginine-induced enzyme release. Whereas poly-L-arginine alone induces little superoxide generation in rabbit PMNs, there is a synergistic enhancement of superoxide production when GTP gamma S and poly-L-arginine are present together. Guanine nucleotides apparently have a modulating effect on the actions of poly-L-arginine on the PMN, but the nature of this effect remains to be determined.

Circadian rhythm of the rat suprachiasmatic brain slice is rapidly reset by daytime application of cAMP analogs.

Cellular mechanisms underlying the primary circadian pacemaker in mammals were investigated by isolating rat suprachiasmatic nuclei in brain slices and maintaining them in vitro for up to 3 days. The circadian rhythm of neuronal firing rate was used to assess the phase of the pacemaker. This rhythm was rapidly reset by bath application of cAMP analogs. Moreover, the pacemaker demonstrated circadian sensitivity to analog treatment: the rhythm was advanced by application during the donor's day, but not during the donor's night. These results suggest that cAMP-mediated events may stimulate pacemaker afferents within the SCN or may directly influence the pacemaker mechanism.

Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma X glioma hybrid NG108-15 cells: effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction.

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca2+ concentration [( Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTP gamma S) but not of GDP. GTP gamma S, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTP gamma S into single cells induced increases in [Ca2+]i. These effects of bradykinin and GTP gamma S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with Kd values of 0.80 +/- 0.26 and 9.63 +/- 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.