P2Y₂ receptor activation decreases blood pressure and increases renal Na⁺ excretion

ATP and UTP are endogenous agonists of P2Y(2/4) receptors. To define the in vivo effects of P2Y(2) receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y(2/4) receptor agonist and UTP analog, in wild-type (WT) and P2Y(2) receptor knockout (P2Y(2)-/-) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS45973 (0.1 to 3 mg/kg body wt) dose-dependently decreased blood pressure in WT (maximum response -35 ± 2 mmHg) and to a similar extent in endothelial nitric oxide synthase knockout mice. By contrast, blood pressure increased in P2Y(2)-/- (maximum response +18 ± 1 mmHg) but returned to basal levels within 60 s. Continuous infusion of INS45973 (25 to 750 μg·min(-1)·kg(-1) body wt) dose-dependently increased urinary excretion of Na(+) in WT (maximum response +46 ± 15%) but reduced Na(+) excretion in P2Y(2)-/- (maximum responses of -45 ± 15%) mice. In renal clearance experiments, INS45973 did not affect glomerular filtration rate but lowered blood pressure and increased fractional excretion of fluid, Na(+), and K(+) in WT relative to P2Y(2)-/- mice. The blood pressure responses to INS45973 are consistent with P2Y(2) receptor-mediated NO-independent vasodilation and implicate responses to endothelium-derived hyperpolarizing factor, and P2Y(2) receptor-independent vasoconstriction, probably via activation of P2Y(4) receptors on smooth muscle. Systemic activation of P2Y(2) receptors thus lowers blood pressure and inhibits renal Na(+) reabsorption, effects suggesting the potential utility of P2Y(2) agonism in the treatment of hypertension.     

Extracellular nucleotides regulate CCL20 release from human primary airway epithelial cells, monocytes and monocyte-derived dendritic cells

Extracellular nucleotides regulate ion transport and mucociliary clearance in human airway epithelial cells (HAECs) via the activation of P2 receptors, especially P2Y(2). Therefore, P2Y(2) receptor agonists represent potential pharmacotherapeutic agents to treat cystic fibrosis (CF). Nucleotides also modulate inflammatory properties of immune cells like dendritic cells (DCs), which play an important role in mucosal immunity. Using DNA-microarray experiments, quantitative RT-PCR and cytokine measurements, we show here that UTP up-regulated approximately 2- to 3-fold the antimicrobial chemokine CCL20 expression and release in primary HAECs cultured on permeable supports at an air-liquid interface (ALI). Both P2Y(2) (ATPgammaS, UTP, INS365) and P2Y(6) (UDP, INS48823) agonists increased CCL20 release. UTP-induced CCL20 release was insensitive to NF-kappaB pathway inhibitors but sensitive to inhibitors of ERK1/2 and p38/MAPK pathways. Furthermore, UTP had no effect on interleukin-(IL)-8 release and reduced the release of both CCL20 and IL-8 induced by TNF-alpha and LPS. Accordingly, UTP reduced the capacity of basolateral supernatants of HAECs treated with TNF-alpha or LPS to induce the chemoattraction of both CD4(+) T lymphocytes and neutrophils. In addition, we show that, in monocyte-derived DCs, ATPgammaS, and UDP but not UTP/INS365-stimulated CCL20 release. Likewise, UDP but not ATPgammaS was also able to increase CCL20 release from monocytes. Pharmacological experiments suggested an involvement of P2Y(11) or P2Y(6) receptors through NF-kappaB, ERK1/2, and p38/MAPK pathways. Altogether, our data demonstrate that nucleotides may modulate chemokine release and leukocyte recruitment in inflamed airways by acting on both epithelial and immune cells. Our results could be relevant for further clinical investigations in CF.     

Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure.

We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor mRNA-expression was quantified in hearts from CHF rats and compared to sham-operated rats. Furthermore, the presence of receptor mRNA was studied in the myocardium from patients with heart failure. In the sham operated rats the G-protein coupled P2Y-receptors were expressed at a higher level than the ligand gated ion-channel receptor (P2X1). Among the P2Y-receptors the P2Y6-receptor was most abundantly expressed (P2Y6 > P2Y1 > P2Y2 = P2Y4 > P2X1). A prominent change was seen for the P2X1- and P2Y2-receptor mRNA levels which were increased 2.7-fold and 4.7-fold respectively in the myocardium from the left ventricle of CHF-rats. In contrast, the P2Y1-, P2Y4- and P2Y6-receptor mRNA levels were not significantly altered in CHF rats. In human myocard the P2X1-, P2Y1-, P2Y2-, P2Y6- and P2Y11-receptors were detected by RT-PCR in both right and left atria and ventricles, while the P2Y4-receptor band was weak or absent. In conclusion, most of the studied P2-receptors were expressed in both rat and human hearts. Furthermore, the P2X1- and P2Y2-receptor mRNA were upregulated in CHF, suggesting a pathophysiological role for these receptors in the development of heart failure.     

Current knowledge on the nucleotide agonists for the P2Y2 receptor

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y2 receptors are widely expressed and play important roles in multiple functionalities. Diquafosol tetrasodium, known as INS365, which was the first P2Y2 receptor agonists that had been approved in April 2010 and launched in Japan by Santen Pharmaceuticals. Besides, a series of similar agonists for the P2Y2 receptor are undergoing development to cure different diseases related to the P2Y2 receptor. This article illustrated the structure and functions of the P2Y2 receptor and focused on several kinds of agonists about their molecular structures, research progress and chemical synthesis methods. Last but not the least, we summarized the structures-activity relationship (SAR) of agonists for the P2Y2 receptor and expected more efficient agonists for the P2Y2 receptor.     

Epithelial and endothelial flux after bypass in dogs: effect of positive end-expiratory pressure

Cardiopulmonary bypass (CPB) causes lung injury that occasionally progresses to the adult respiratory distress syndrome (ARDS). We measured the effect of 10 cmH2O of positive end-expiratory pressure (PEEP) on small solute and protein flux in dogs 1 wk before and 2 h after the completion of CPB. As an index of alveolar epithelial permeability, the clearance from lung to blood of inhaled technetium-99m-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) was measured. To assess microvascular endothelial integrity, the rate of accumulation in the lung interstitium of intravascular 113mIn-transferrin was measured. The clearance half time (t 1/2) for 99mTc-DTPA in the study dogs declined from 18.8 +/- 1.9 min (mean +/- SE) at base line to 9.4 +/- 2.0 min during PEEP (P less than 0.05). Two hours after CPB, the t 1/2 was 8.1 +/- 1.6 min at base line and unchanged during PEEP. The 113mIn-transferrin rate of accumulation was unchanged by PEEP before CPB. After CPB, the index was 3.25 +/- 0.95 slope/min X 10(-3) (P less than 0.05). Of the five dogs with a significant slope, four showed a decrease in microvascular flux during PEEP, although for the group the mean change in slope was not significant (P = 0.10). We conclude that the application of PEEP does not increase 99mTc-DTPA clearance in lungs already injured by CPB, and may actually decrease the apparent microvascular protein flux in some cases.     

Saline aerosol bolus dispersion. I. The effect of acinar airway alteration.

We explored the possibility of using a saline aerosol for bolus dispersion measurements to detect peripheral airway alterations in smokers. Indexes of ventilation inhomogeneity in conductive (S(cond)) and acinar (S(acin)) lung zones, as derived from the multiple-breath N(2) washout (Verbanck S, Schuermans D, Van Muylem A, Noppen M, Paiva M, and Vincken W, J Appl Physiol 83: 1807-1816, 1997), were also measured. The saline bolus test consisted of inhaling 60-ml saline aerosol boluses to different volumetric lung depths (VLD) in the 1.1 liter volume above functional residual capacity. In the never-smoker group (n = 12), saline boluses showed bolus dispersion values consistent with normal values reported in the literature for 0.5- to 1-microm aerosols. In the smoker group (n = 12; 28 +/- 9 pack years, mean +/- SD), significant increases were seen on dispersion and skew of the most peripherally inhaled saline boluses (VLD = 800 ml; P < 0.05) as well as on S(acin) (P = 0.007) with respect to never-smokers. Shallow inhaled boluses (VLD = 200 ml) and S(cond) did not reveal any significant differences between smokers and never-smokers. This study shows the consistent response of two conceptually independent tests, in which both saline aerosol and gas-derived indexes point to a heterogeneous distribution of smoking-induced structural alterations in the lung periphery.     

Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP(3)-independent Ca(2+)-influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca(2+)-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(Asn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca(2+)-independent cascade.     

Amiloride impairs lung water clearance in newborn guinea pigs

To determine whether epithelial ion transport is physiologically important for lung water clearance after birth, the sodium transport inhibitor amiloride or its vehicle saline was given intratracheally to newborn full-term guinea pigs before the first breath. Guinea pigs given saline intratracheally breathed normally and had arterial O2 saturations (SaO2) greater than 94%. In contrast, guinea pigs that had an estimated 10(-4) M intra-alveolar concentration of amiloride had chest wall retractions and 88 +/- 3.6% (SD) SaO2 (P less than 0.01). Extravascular lung water (EVLW) per gram of dry lung weight 4 h after birth was significantly greater in newborns that received amiloride (8.3 +/- 1.1, n = 5) than in those that received saline (5.6 +/- 0.9, n = 7, P less than 0.01). The degree of perivascular fluid cuffing at 25 cmH2O inflation was quantitatively similar in amiloride- and saline-treated animals. The effect of amiloride was dose dependent. Intratracheal amiloride did not affect EVLW in 9-day-old guinea pigs. This study demonstrates that intratracheal amiloride before the first breath results in respiratory distress, hypoxemia, and an abnormally high EVLW. Epithelial sodium transport contributes normal lung liquid clearance after birth.     

A Pilot Study to Examine the Effect of Chronic Treatment with Immunosuppressive Drugs on Mucociliary Clearance in a Vagotomized Murine Model

Background

Previously, we have demonstrated that mucociliary clearance (MCC) is diminished within the first months after surgery in lung transplant patients and the explanation for the reduction in MCC is unknown. We hypothesized that chronic treatment with a commonly prescribed regimen of immunosuppressive drugs significantly impairs MCC. We tested this hypothesis in a murine model of lung transplantation.

Methods

Fifteen C57BL/6 mice underwent vagotomy on the right side to simulate denervation associated with lung transplantation in humans. For 6 days, seven mice (controls) were intraperitoneally injected with three 100 µL doses of phosphate buffered saline and eight mice (immunosuppressed) were injected with three 100 µL injections of tacrolimus (1 mg/kg), mycophenolate mofetil (30 mg/kg), and prednisone (2 mg/kg) once daily. Then, mice inhaled the radioisotope ^99mtechnetium and underwent gamma camera imaging of their lungs for 6.5 hrs. Counts in the right lung at 1–1.5 hrs and at 6–6.5 hrs were first background-corrected and then decay-corrected to time 0 counts. Decay-corrected counts were then divided by time 0 counts. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain percent removed by mucociliary clearance.

Results

Although there was a slowing of MCC at 1–1.5 hrs for the immunosuppressed mice, there was no statistical difference in MCC measured at 1–1.5 hrs for the two groups of mice. At 6–6.5 hrs, MCC was significantly slower in the immunosuppressed mice, compared to controls, with 7.78±5.9% cleared versus 23.01±11.7% cleared, respectively (p = 0.006).

Conclusions

These preliminary results suggest that chronic treatment with immunosuppressive medications significantly slows MCC in vagotomized C57BL/6 mice. These findings could shed light on why MCC is reduced in lung transplant patients whose lungs are denervated during surgery and who are chronically treated with immunosuppressive drugs post surgery.     

Nucleotides released from Aβ₁₋₄₂ -treated microglial cells increase cell migration and Aβ₁₋₄₂ uptake through P2Y₂ receptor activation

Amyloid β-protein (Aβ) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aβ. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aβ. Treatment of mouse primary microglial cells with fibrillar (fAβ(1-42) ) and oligomeric (oAβ(1-42) ) Aβ(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAβ(1-42) and oAβ(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAβ(1-42) and oAβ(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aβ(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aβ(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aβ(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aβ phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aβ(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aβ clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.     

ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Methylcholine-activated eccrine sweat gland density and output as a function of age

The purpose of this investigation was to examine eccrine sweat gland responsiveness to intradermal injections of methylcholine (MCh) across three age groups of men [young (Y) = 22-24; middle (M) = 33-40; older (O) = 58-67 yr old, n = 5 per group]. Subjects were matched with respect to maximum O2 consumption, body size, and body composition, and were thoroughly heat acclimated before participation. Randomly ordered concentrations of acetyl-beta-methylcholine chloride ranging from 0% (saline) to 0.1% (5 x 10(-3) M) were injected into the skin of the dorsal thigh in a thermoneutral environment, and activated sweat glands were photographed at 30-s intervals for the next 8 min. Density of MCh-activated glands was independent of both age and [MCh] (e.g., 2 min after injection of 5 x 10(-3) M [MCh]: Y = 45 +/- 7, M = 46 +/- 12, O = 42 +/- 5 glands/cm2). However, sweat gland output (SGO) per active gland was significantly lower for the O group and failed to increase with increasing [MCh] above 5 x 10(-4) M. When MCh (5 x 10(-3) M) was injected after 1 h of exercise in the heat, higher SGO's were elicited in each group; however, the SGO of the O group was again significantly lower than that of the Y group (91 +/- 11 vs. 39 +/- 4 ng/gland, P less than 0.02) with the M group intermediate (69 +/- 11 nl/gland; 2 min postinjection data).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alveolar and lung liquid clearance in anesthetized rabbits

Alveolar and lung liquid clearance were studied over 8 h in intact anesthetized ventilated rabbits by instillation of either isosmolar Ringer lactate (2 ml/kg) or autologous plasma (2 or 3 ml/kg) into one lower lobe. The half time for lung liquid clearance of the isosmolar Ringer lactate was 3.3 h and that for plasma clearance was 6 h. In the plasma experiments, the alveolar protein concentration after 1 h was 5.2 +/- 0.8 g/dl, which was significantly greater than the initial instilled protein concentration of 4.3 +/- 0.7 g/dl (P less than 0.05). Thus alveolar protein concentration increased by 21 +/- 12% over 1 h, which matched clearance from the entire lung of 19 +/- 11% of the instilled volume. Overall the rate of alveolar and lung liquid clearance in rabbits was significantly faster than in prior studies in dogs and sheep. The fast alveolar liquid clearance rate in rabbits was not due to higher endogenous catecholamine release, because intravenous and alveolar (5 x 10(-5) M) propranolol did not slow the clearance. Also, beta-adrenergic therapy with alveolar terbutaline (10(-5) or 10(-4) M) did not increase the alveolar or lung liquid clearance rates. Phloridzin (10(-3) M) did not slow alveolar liquid clearance. However, amiloride (10(-4) M) inhibited 75% of the basal alveolar liquid clearance in rabbits, thus providing evidence that alveolar liquid clearance in rabbits depends primarily on sodium-dependent transport. This rabbit study provides further evidence for important species differences in the basal rates of alveolar liquid and solute clearance as well as the response to beta-adrenergic agonists and ion transport inhibitors.     

Lung epithelial permeability to aerosolized solutes: relation to position

The lung epithelial permeability to inhaled solutes is primarily attributed to the degree of distension of the interepithelial junctions and thus of the alveolar volume. To assess this hypothesis, a submicronic aerosol of technetium-99m-labeled diethylenetriamine pentaacetate (99mTc-DTPA) was inhaled by eight normal subjects in left lateral decubitus (LLD). The regional lung clearance of 99mTc-DTPA was measured in LLD, then in right lateral decubitus (RLD) to reverse the relative distension of the alveoli. Although in LLD the deposition of the aerosol is the greatest in the gravity-dependent regions of the left lung, their 99mTc-DTPA clearances are significantly lower than those of the nondependent regions of the right lung (0.7 +/- 0.3 vs. 2 +/- 0.8%/min, P less than 0.001). In RLD, these regions placed in opposite positions significantly reversed their clearances (1.6 +/- 0.8 vs. 0.6 +/- 0.2%/min, P less than 0.001). Results indicate in lateral decubitus a gravity gradient of 99mTc-DTPA clearances independent of the aerosol deposition. This gradient of epithelial permeability to solutes appears to be influenced by the gradient of alveolar volume.     

Simulated first-phase insulin release using Humulin or insulin analog HIM2 is associated with prolonged improvement in postprandial glycemia

We examined the extent to which priming the liver with a pulse of Humulin or the insulin analog hexyl-insulin monoconjugate 2 (HIM2) reduces postprandial hyperglycemia. Somatostatin (0.5 microg.kg(-1).min(-1)) was given with basal intraportal insulin and glucagon for 4.5 h into three groups of 42-h-fasted conscious dogs. From 0-5 min, group 1 (BI, n = 6) received saline, group 2 (HI, n = 6) received a Humulin pulse (10 mU.kg(-1).min(-1)), and group 3 (HIM2, n = 6) received a HIM2 pulse (10 mU.kg(-1).min(-1)). Duodenal glucose was infused (5.0 mg.kg(-1).min(-1)) from 15 to 270 min. Arterial insulin in BI remained basal (6 +/- 1 microU/ml) and peaked at 52 +/- 15 (HI) and 164 +/- 44 microU/ml (HIM2) and returned to baseline by 30 and 60 min, respectively. Arterial plasma glucose plateaued at 265 +/- 20, 214 +/- 15, and 193 +/- 14 mg/dl in BI, HI, and HIM2. Glucose absorption was similar in all groups. Significant net hepatic glucose uptake occurred at 85, 55, and 25 min in BI, HI, and HIM2, respectively. Nonhepatic glucose clearance at 270 min differed among groups (BI, HI, HIM2): 0.62 +/- 0.11, 0.76 +/- 0.26, and 1.61 +/- 0.29 ml.kg(-1).min(-1) (P < 0.05). A brief (5-min) insulin pulse improved postprandial glycemia, stimulating hepatic glucose uptake and prolonging enhancement of nonhepatic glucose clearance. HIM2 was more effective than Humulin, perhaps because its lowered clearance caused higher levels at the liver and periphery and its biological activity was not reduced proportionally to its decreased clearance.     

Stimulatory effect of PG-KII, an NK3 tachykinin receptor agonist, on isolated pancreatic acini: species-related differences

More information is needed on the physiological role of the tachykinins (TKs), especially neurokinin3-receptor (NK3) agonists, in the pancreas. In this paper we investigated and compared the effect of PG-KII (10(-9) to 10(-6) M), a natural NK3-receptor agonist, with that of the known secretagogues substance P (10(-9) to 10(-6)M), caerulein (10(-11) to 10(-8) M) and carbachol (10(-8) to 10(-5) M), on amylase secretion from dispersed pancreatic acini of the guinea pig and rat. PG-KII (10(-7) M) significantly increased basal amylase release from guinea pig pancreatic acini (from 5.4+/-0.9% to 11.3+/-0.5%, P < 0.05) but left basal release in the rat unchanged (6.5+/-0.5%). The stimulant effect of PG-KII on guinea pig acini was significantly reduced by the NK3-receptor antagonist, SR 142801 (5 x 10(-7) M), and left unchanged by the NK1-receptor antagonist, SR 140333 (5 x 10(-7) M). Conversely, substance P (10(-7) M) significantly stimulated amylase secretion from rat and guinea pig acini (12.6+/-0.6% and 12.1+/-0.7%, P < 0.05). This stimulated effect of substance P was antagonized by the NK1--receptor antagonist (5 x 10(-7) M), but not by the NK3-receptor antagonist (5 x 10(-7) M). The PG-KII- and substance P-evoked maximal responses were lower than those evoked by caerulein (10(-9) M) (guinea pig, 19.1+/-1.3%; rat, 1802+/-0.9%, P < 0.01) and carbachol (10(-5) M) (guinea pig, 23.3+/-1.2%; rat, 24.0+/-1.1%, P < 0.01). The inhibitors of phospholipase C U-73122 (10(-5) M), phospholipase A2 quinacrine (10(-5)M), and protein tyrosine kinase genistein (10(-4) M), partly but significantly inhibited PG-KII, as well as carbachol-stimulated amylase release. Coincubation of PG-KII 10(-7) M with submaximal doses of caerulein (10(-11) to 10(-10) M) and carbachol (10(-7) to 10(-6) M) had an additive effect on amylase release. Pre-incubation with PG-KII (10(-7) M) for 30 min significantly reduced the subsequent amylase response to PG-KII, whereas pre-incubation with caerulein 10(-10) M or carbachol 10(-6) M did not. These findings suggest that PG-KII directly contributes to pancreatic exocrine secretion by interacting with acinar NK3 receptors of the guinea pig but not of the rat. PG-KII signal transduction involves the intracellular phospholipase C, phospholipase A2 and protein tyrosine kinase pathways. The NK3 receptor system cooperates with the other known secretagogues in regulating guinea pig exocrine pancreatic secretion and undergoes rapid homologous desensitization.     

Maximal rowing has an acute effect on the blood-gas barrier in elite athletes.

The purpose of the study was to evaluate the effects of maximal exercise on the integrity of the alveolar epithelial membrane using the clearance rate of aerosolized 99mTc-labeled diethylenetriaminepentaacetic acid as an index for the permeability of the lung blood-gas barrier. Ten elite rowers (24.3 +/- 4.6 yr of age) completed two 20-min pulmonary clearance measurements immediately after and 2 h after 6 min of all-out rowing (initial and late, respectively). All subjects participated in resting control measurements on a separate day. For each 20-min measurement, lung clearance was calculated for 0-7 and 10-20 min. Furthermore, scintigrams were processed from the initial and late measurements of diethylenetriaminepentaacetic acid clearance. Compared with control levels, the pulmonary clearance measurement after rowing was increased from 1.2 +/- 0.5 to 2.4 +/- 1.0%/min (SD) at 0-7 min (P < 0.01) and from 0.8 +/- 0.3 to 1.5 +/- 0.4%/min at 10-20 min (P < 0.0005), returning to resting levels within 2 h. In 6 of 10 subjects, ventilation distribution on the lung scintigrams was inhomogeneous at the initial measurement. The study demonstrates an acute increased pulmonary clearance after maximal rowing. The ventilation defects identified on the lung scintigrams may represent transient interstitial edema secondary to increased blood-gas barrier permeability induced by mechanical stress.     

Extracellular ATP-mediated signaling for survival in hyperoxia-induced oxidative stress

Respiratory failure is a serious consequence of lung cell injury caused by treatment with high inhaled oxygen concentrations. Human lung microvascular endothelial cells (HLMVEC) are a principal target of hyperoxic injury (hyperoxia). Cell stress can cause release of ATP, and this extracellular nucleotide can activate purinoreceptors and mediate responses essential for survival. In this investigation, exposure of endothelial cells to an oxidative stress, hyperoxia, caused rapid but transient ATP release (20.03 +/- 2.00 nm/10(6) cells in 95% O(2) versus 0.08 +/- 0.01 nm/10(6) cells in 21% O2 at 30 min) into the extracellular milieu without a concomitant change in intracellular ATP. Endogenously produced extracellular ATP-enhanced mTOR-dependent uptake of glucose (3467 +/- 102 cpm/mg protein in 95% oxygen versus 2100 +/- 112 cpm/mg protein in control). Extracellular addition of ATP-activated important cell survival proteins like PI 3-kinase and extracellular-regulated kinase (ERK-1/2). These events were mediated primarily by P2Y receptors, specifically the P2Y2 and/or P2Y6 subclass of receptors. Extracellular ATP was required for the survival of HLMVEC in hyperoxia (55 +/- 10% surviving cells with extracellular ATP scavengers [apyrase + adenosine deaminase] versus 95 +/- 12% surviving cells without ATP scavengers at 4 d of hyperoxia). Incubation with ATP scavengers abolished ATP-dependent ERK phosphorylation stimulated by hyperoxia. Further, ERK activation also was found to be important for cell survival in hyperoxia, as treatment with PD98059 enhanced hyperoxia-mediated cell death. These findings demonstrate that ATP release and subsequent ATP-mediated signaling events are vital for survival of HLMVEC in hyperoxia.     

Prostaglandins potentiate U-46619-induced pulmonary microvascular dysfunction.

The induction of cyclooxygenase is an important event in the pathophysiology of acute lung injury. The purpose of this study was to examine the synergistic effects of various cyclooxygenase products (PGE(2), PGI(2), PGF(2alpha)) on thromboxane A(2) (TxA(2))-mediated pulmonary microvascular dysfunction. The lungs of Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing indomethacin and PGE(2) (5 x 10(-8) to 1 x 10(-7) M), PGF(2alpha) (7 x 10(-9) to 5 x 10(-6) M), or PGI(2) (5 x 10(-8) to 2 x 10(-5) M). The TxA(2)-receptor agonist U-46619 (7 x 10(-8) M) was then added to the perfusate, and then the capillary filtration coefficient (K(f)), pulmonary arterial pressure (Ppa), and total pulmonary vascular resistance (RT) were determined. The K(f) of lungs perfused with U-46619 was twice that of lungs perfused with buffer alone (P = 0.05). The presence of PGE(2), PGF(2alpha), and PGI(2) within the perfusate of lungs exposed to U-46619 caused 118, 65, and 68% increases in K(f), respectively, over that of lungs perfused with U-46619 alone (P < 0.03). The RT of lungs perfused with PGE(2) + U-46619 was approximately 30% greater than that of lungs exposed to either U-46619 (P < 0.02) or PGE(2) (P < 0.01) alone. When paired measurements of RT taken before and then 15 min after the addition of U-46619 were compared, PGI(2) was found to attenuate U-46619-induced increases in RT (P < 0.01). These data suggest that PGE(2), PGI(2), and PGF(2alpha) potentiate the effects of TxA(2)-receptor activation on pulmonary microvascular permeability.     

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.