AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 286: H1027-H1033, 2004. First published October 23, 2003; doi:10.1152/ajpheart.00630.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/3/H1027    most recent
00630.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nan, Y.-S.
Right arrow Articles by Ishikawa, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nan, Y.-S.
Right arrow Articles by Ishikawa, N.

Neuropeptide Y enhances permeability across a rat aortic endothelial cell monolayer

Yong-Shan Nan,1 Guo-Gang Feng,2 Yoshihiro Hotta,2 Kimitoshi Nishiwaki,1 Yasuhiro Shimada,1 Atsuko Ishikawa,2 Nakako Kurimoto,3 Tatsuro Shigei,2 and Naohisa Ishikawa2

1Department of Anesthesiology, Nagoya University School of Medicine, Nagoya 480-1195; and Departments of 2Pharmacology and 3Anesthesiology, Aichi Medical University School of Medicine, Aichi 480-1195, Japan

Submitted 3 July 2003 ; accepted in final form 22 October 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, in vivo studies showed that neuropeptide Y (NPY) elevates vascular permeability in isolated lung perfusion preparations, possibly through binding to the NPY Y3 receptor. The present study used monolayers in a double-chamber culture method under conditions of normoxia (5% CO2-20% O2-75% N2) or hypoxia (5% CO2-5% O2-90% N2) to test the hypothesis that NPY directly affects rat aortic endothelial cells (RAECs). RAECs were cultured on the base of the upper chamber, into which FITC-labeled albumin was introduced, and permeation into the lower chamber was measured. The RAEC monolayer was treated with 10–8–3 x 10–7 M NPY for 2 h in normoxia or hypoxia. In hypoxia, NPY concentration dependently increased the permeability of the RAEC monolayer, whereas in normoxia no significant change was observed. Peptide YY, NPY Y1, and NPY Y2 receptor agonists and NPY Y1 receptor antagonist exerted no significant effects under hypoxic conditions. NPY-(18–36), an NPY Y3 receptor antagonist, elicited an inhibitory action on the NPY-induced increase in monolayer permeability. Furthermore, neither N-monomethyl-L-arginine, a nitric oxide synthase inhibitor, the bradykinin B2 receptor antagonist FK-3657, nor the vascular endothelial growth factor receptor-coupled tyrosine kinase inhibitor tyrphostin SU-1498, injected into the medium of the upper chamber, affected the NPY-induced permeability changes under hypoxic conditions. The results suggest that the NPY-induced increase in permeability across the RAEC monolayer is closely related to low O2 tension, possibly mediated by direct action on the NPY Y3 receptor expressed on the endothelial cell membrane. Furthermore, this NPY-induced increase is not likely due to nitric oxide, bradykinin, or vascular endothelial growth factor.

peptide YY; hypoxia

NEUROPEPTIDE Y (NPY) is a 36-amino acid neurotransmitter that is widely distributed throughout the central and peripheral nervous system (9, 39). In the latter, NPY acts as a coneurotransmitter with norepinephrine at the sympathetic nerve terminals, being released when the sympathetic nerves are highly excited, as in the case of strong stress. In addition to central actions in control of food intake (2), cerebrocortical excitability (42), and integration of emotional behavior (13), NPY has been implicated in peripheral functions in the cardiovascular system (36) and neurogenic pulmonary edema (14, 35).

Among the six different NPY receptors, Y1–Y6, only the NPY Y3 receptor has not been cloned (20, 25). The NPY Y3 receptor is pharmacologically characterized by its inability to be activated by peptide YY (PYY) (25), thus essentially differing from the other five receptor subtypes. In the course of studies on mechanisms of neurogenic pulmonary edema, Hirabayashi et al. (14) used isolated lung perfusion preparations to demonstrate that NPY dose dependently increased capillary filtration coefficients in the rat pulmonary vascular system. They further established that the elevation of vascular permeability was mediated by the NPY Y3 receptor (14). The Y3 receptor is very sparsely distributed, and few reports have appeared concerning its functions, for example, in the rat cardiopulmonary system (11) and in bovine chromaffin cells (41). Noll et al. (32) reported that NPY reduced macromolecule permeability across coronary endothelial monolayers through modulation of cAMP-dependent signal transduction but did not identify the NPY receptor subtype. There were discrepancies between the permeability results of Noll et al. and those of Hirabayashi et al., but their methods were different: cultured endothelial cells were used in the former, and an in vivo preparation was used in the latter. Usually, with isolated lung perfusion preparations, the lungs are inflated and maintained without respiratory movement and, therefore, might be under hypoxic conditions. In contrast, the experiments reported by Noll et al. were performed under normoxic conditions.

The endothelial cell layer forms a permeability barrier between circulating blood and the underlying vascular tissue, restricting fluid and material flow across the vascular wall. It is well known that the permeability of the endothelial barrier is increased in hypoxia, for example, in human microvascular endothelial cells and rat blood-brain barrier endothelial cells (33, 34). Hypoxia may release some mediators such as vascular endothelial growth factor (VEGF) (29) and nitric oxide (NO) (34), both of which increase vascular permeability. In human umbilical vein endothelial cells (HUVECs) (29), these effects did not appear until 24–48 h after exposure to 5% O2 hypoxia. These vascular responses to hypoxia contribute to acute respiratory distress syndrome (27) and pulmonary embolism and ischemia-reperfusion injury (4). Previous reports demonstrated that acute hypoxia or ischemia-reperfusion caused release of circulating NPY (40), possibly from sympathetic nerves or other extraneuronal cells such as platelets (28) and endothelial elements (21), increasing NPY concentration in the plasma of sheep fetus (7). In this context, we hypothesize that hypoxia-induced elevation of vascular permeability may also be affected by another possible permeability mediator, NPY. Before examining such a hypothesis, in the present study, using a monolayer of rat aortic endothelial cells (RAECs), we evaluated the effects of NPY on large molecule permeability in normoxia and hypoxia. Furthermore, the study was undertaken to clarify a possible NPY receptor subtype participating in the permeability response of RAEC monolayers to NPY and to examine whether permeability mediators such as VEGF, NO, bradykinin, and prostaglandins are associated with NPY activity.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Throughout the experiments, all animals were handled in accordance with the guidelines for animal experimentation set by the Japanese Association for Laboratory Animal Science, and the protocol used in the present study was approved by the Animal Care Committee of Aichi Medical University.

Isolation and culture of endothelial cells. RAECs were isolated from male Wistar rats (150–200 g, 7–9 wk old) and cultured according to the methods of Suh et al. (38). Briefly, rats were anesthetized with ketamine (50 mg/kg body wt im) and pentobarbital sodium (25 mg/kg body wt ip), and their aortas were removed and placed in phosphate-buffered saline (PBS, without Ca2+ or Mg2+). The vessels were cleaned, opened longitudinally, cut into two or three small pieces, and placed with their intimal side down on Matrigel-coated plates in growth medium (GM). GM contained 10% fetal calf serum, 75 µg/ml endothelial cell growth supplement, 10 U/ml heparin, 100 U/ml penicillin-streptomycin, 1% L-glutamine, and 100 µM MEM nonessential amino acids in DMEM. After 4–7 days, the pieces were removed and the cells were harvested. When assessed with trypan blue, viability of the primary cultured cells was >90%.

Establishment of endothelial cell monolayers for permeability assays. The incubation culture plates were composed of two chambers. The base of the upper chamber was a sieve with a 3-µm pore size (Chemotaxicell, Kurabo, Osaka, Japan). We used 24-well microplates for the lower chamber. Before use, the upper chamber plate was coated with 50 µl of 50 µg/ml collagen IV and left to dry overnight in a laminar airflow cabinet. The chambers were then sterilized by rinses with 70% ethanol and allowed to dry. Trypsin-EDTA was used to detach RAECs from the culture plates, and the cells were washed once with fresh GM and seeded at a density of 2 x 105 cells/well in 200 µl of GM. They were incubated at 37°C in 5% CO2-95% air for 4 days, during which time GM (300 µl) was changed every day.

Measurement of endothelial permeability. Endothelial monolayer permeability was assessed as the filtration velocity of FITC-labeled albumin from the upper to the lower chamber, as previously described (23). Into the upper chamber, 300 µl of 1% FITC-labeled albumin GM were added; 1 ml of GM containing 1% BSA was introduced into the lower chamber. The cells were incubated for 2 or 24 h in normoxia (5% CO2-20% O2-75% N2) or hypoxia (5% CO2-5% O2-90% N2). Soon after administration of FITC-labeled albumin, NPY, PYY, [Leu31,Pro34]-NPY-(13–36) (an NPY Y1 receptor agonist), NPY-(13–36) (an NPY Y2 receptor agonist), bradykinin, or histamine was injected into the upper chamber medium, and the cells were incubated for 2–6 h. From the lower chamber, 100-µl aliquots were aspirated and diluted 1:5 with PBS before the concentration of FITC-labeled albumin was measured, as an index of monolayer permeability. The amount of FITC-labeled albumin was determined using a fluorescent spectrophotometer (Fluoroskan Ascent FL, Labsystems), with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The monolayer was treated with 10–5 M NPY-(18–36), an NPY Y3 receptor antagonist, 10 min before incubation with 3 x 10–7 M NPY. The monolayer was also pretreated with 3 x 10–5 M diphenylacetyl-D-arg-4-hydroxybenzylamide (BIBP-3226), an NPY Y1 receptor antagonist; 10–5 M N-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor; 10–5 M FK-3657, a bradykinin B2 receptor antagonist (15); 10–6 M indomethacin, a cyclooxygenase inhibitor; or 10–6 M tyrphostin SU-1498, a VEGF (KDR) receptor-coupled tyrosine kinase inhibitor. To examine the efficiency of FK-3657 and tyrphostin SU-1498, pretreatment with either agent was followed by 10–5 M bradykinin and 10 ng/ml VEGF, respectively.

Measurement of intracellular cAMP. Intracellular cAMP was evaluated using a cAMP fluorescence polarization Biotrak immunoassay kit according to the manufacturer's instructions. Briefly, cells (50 µl) were incubated on 96-well microplates at a density of 106 cells/ml overnight at 37°C in 5% CO2-95% air. After administration of NPY into the wells, the cells were maintained for 2 h in normoxia or hypoxia. Thereafter, cells were lysed with 50 µl of cell lysis buffer and treated with 50 µl of rabbit anti-cAMP serum and then with Cy3B-cAMP conjugate. The antibody-bound Cy3B-cAMP conjugate elicits polarization, which may be measurable with the fluorescent spectrophotometer at an excitation wavelength of 535 nm and an emission wavelength of 590 nm. The content of cAMP in the cultured cells competing with Cy3B-cAMP conjugate in binding to the antibody was obtained with a standard curve.

Measurement of intracellular Ca2+ concentration in RAECs. RAECs were placed on 96-well culture plates coated with poly-L-lysine at a density of ~2 x 105 cells/well and incubated in 5% CO2-95% air for 18 h. The cells were washed three times with Tyrode solution before being loaded with 50 µl of 20 µM fluo 3-AM at 25°C for 1 h, and then excess dye was removed by three washes with Tyrode solution. Thereafter, baseline fluorescence (Fb) with 50 µl of Tyrode solution was measured in each well. Then, 50 µl of 3 x 10–7 M NPY, 10–6 M PYY, or 50 µl of Tyrode solution (as control) were applied to the cells. We calculated {Delta}F as the difference between Fb and measured fluorescence. Fmax and Fmin were calculated by subtracting Fb from signals obtained by addition of 20 µl of 1% NP-40 and 15 µl of 0.1 M EGTA to the medium 10 min before the measurement. The Ca2+ concentration for each well was calculated according to the following formula: Ca2+ (nM) = Kd x ({Delta}F – Fmin)/(Fmax – {Delta}F), where the dissociation constant (Kd) was 390 nM for fluo 3-AM.

Materials. Matrigel-coated plates were purchased from Becton-Dickinson (Bedford, MA); DMEM, fetal calf serum, trypsin-EDTA, penicillin-streptomycin, L-glutamine, and MEM nonessential amino acid solution from GIBCO Life Technologies (Eggenstein, Germany); NPY-(18–36), endothelial cell growth serum, trypan blue, collagen IV, indomethacin, and FITC-labeled albumin from Sigma (St. Louis, MO); L-NMMA, heparin, BSA, fluo 3-AM, and NP-40 from Wako (Osaka, Japan); Chemotaxicell from Kurabo (Osaka, Japan); 96- and 24-well cell culture plates and poly-L-lysine from Becton-Dickinson Biosciences (Franklin Lakes, NJ); NPY, PYY, [Leu31,Pro34]-NPY-(13–36), BIBP-3226, and NPY-(13–36) from Bachem (Basel, Switzerland); FK-3657 from Fujisawa Pharmaceutical (Osaka, Japan); tyrphostin SU-1498 from LC Laboratories (Boston, MA); and cAMP fluorescence polarization Biotrak immunoassay kit from Amersham Biosciences (Buchler, Germany). Tyrode solution was composed of 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0.2 mM NaH2PO4, 12 mM NaHCO3, and 5.5 mM glucose. To make the stock solution of BIBP-3226, we dissolved BIBP-3226 in DMSO and diluted this solution with 0.1% PBS. A preliminary study showed that 0.1% DMSO did not affect RAEC monolayer permeability.

Statistical analysis. Differences between means were examined for significance with analysis of variance, unless indicated in paired t-tests. Statistical significance was evaluated by Scheffé's method (37) at a level of 0.05, with values expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of NPY on RAEC monolayer permeability. In normoxia, FITC-labeled albumin concentration in the lower chamber, permeating across the RAEC monolayer, increased time dependently, even without NPY, from 63.2 ± 11.4 mg/l just before injection of FITC-labeled albumin into the upper chamber (0 h) to 2,843.8 ± 63.9 mg/l at 6 h (Fig. 1A). When NPY was injected into the upper chamber at 3 x 10–7 M, FITC-labeled albumin concentration increased time dependently, with no significant differences in the mean values with and without NPY.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 1. Time trends for change of FITC-labeled albumin concentration in the lower chamber with permeation through the rat aortic endothelial cell (RAEC) monolayer with and without neuropeptide Y (NPY) in normoxia (A) and hypoxia (B). FITC-labeled albumin was added to the upper chamber at 0 h, and absorbance values were measured every 2 h. FITC-labeled albumin concentration leveled off 6 h after treatment with different concentrations of NPY, which was administered into the upper chamber at 0 h. Values are means ± SE; n = 4. *P < 0.05; **P < 0.01 vs. no drug.

 

At 2 h after induction of hypoxia, FITC-labeled albumin concentration in the lower chamber also increased time dependently, even without NPY (Fig. 1B). When NPY was injected into the upper chamber at the highest concentration of 3 x 10–7 M, FITC-labeled albumin concentration 2 and 4 h after injection of FITC-labeled albumin into the upper chamber was 1,976.9 ± 220.1 and 3,073.0 ± 115.2 mg/l, respectively. Both values were significantly greater than the respective values obtained without NPY at 2 and 4 h (P < 0.01 for each). The mean values obtained with 10–7 M NPY, 1,846.0 ± 262.0 mg/l at 2 h (P < 0.05), and 2,810.6 ± 100.9 mg/l (P < 0.01) at 4 h, were also significantly greater than the values obtained without NPY.

In subsequent studies of monolayer permeability, FITC-labeled albumin was measured after 2 h.

Effects of bradykinin and histamine on RAEC monolayer permeability. Bradykinin and histamine elevated FITC-labeled albumin concentration in the lower chamber in normoxia and hypoxia (Fig. 2). In normoxia, 10–5 M bradykinin significantly increased albumin concentration from 406.3 ± 120.5 to 1,595.1 ± 313.1 mg/l (P < 0.05) and 10–5 M histamine increased albumin concentration to 1,588.4 ± 172.0 mg/l (P < 0.05). Bradykinin and histamine were administered into the upper chamber after 2 or 24 h of hypoxia. FITC-labeled albumin concentration in the lower chamber in the presence of 10–5 M bradykinin was 1,863.4 ± 135.7 and 1,874.2 ± 109.0 mg/l after 2 and 24 h of hypoxia, respectively; both values are significantly greater than those obtained in the absence of bradykinin (P < 0.01). There were no significant differences in FITC-labeled albumin concentrations between 2 and 24 h of hypoxia. FITC-labeled albumin concentration in the presence of 10–5 M histamine was 1,390.9 ± 152.5 and 2,188.7 ± 164.1 mg/l after 2 and 24 h of hypoxia, respectively (P < 0.05); both values are significantly greater than those obtained in the absence of histamine (P < 0.05 for the former and P < 0.01 for the latter).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Effects of bradykinin (Brk) and histamine (His) on FITC-labeled albumin concentration in the lower chamber with permeation through RAEC monolayer. Bradykinin or histamine solution, at 10–5 M, was administered into the incubation medium 2 (2h) or 24 h (24h) after induction of hypoxia or in normoxia (20%). Values are means ± SE; n = 4. *P < 0.05; **P < 0.01 vs. no drug. P < 0.05 vs. His(2h).

 

Effects of concentration-response curve for NPY on RAEC monolayer permeability. NPY concentration dependently increased the FITC-labeled albumin concentration in the lower chamber medium (Fig. 3). FITC-labeled albumin concentration in the presence of 10–7 and 3 x 10–7 M NPY after 2 h of hypoxia was 1,846.0 ± 262.0 and 1,976.9 ± 220.1 mg/l, respectively; both values are significantly greater than those obtained in normoxia (P < 0.01 for each). FITC-labeled albumin concentration in the presence of 10–7 and 3 x 10–7 M NPY after 24 h of hypoxia was 1,147.3 ± 204.2 and 1,298.7 ± 120.5 mg/l, respectively; both values are significantly greater than those obtained in normoxia (P < 0.05 for each). In addition, we compared the concentration-response curves obtained at 2 and 24 h. In normoxia, no marked difference between 2 and 24 h was obtained at any concentration of NPY. In hypoxia, FITC-labeled albumin concentration in the presence of 10–7 and 3 x 10–7 M NPY was significantly lower at 24 h than at 2 h (P < 0.05 for each).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. Concentration-response curves for effects of NPY on RAEC monolayer permeability. Absorbance of FITC-labeled albumin in the lower chamber medium was measured 2 h after addition of FITC-labeled albumin into the upper chamber. Values are means ± SE; n = 4. *P < 0.05; **P < 0.01 vs. normoxia; P < 0.05 vs. 24 h in hypoxia.

 

NPY did not affect RAEC monolayer permeability in normoxia, whereas bradykinin and histamine elevated FITC-labeled albumin concentration, even in normoxia. Furthermore, NPY decreased FITC-labeled albumin concentration from 2 to 24 h after induction of hypoxia; histamine increased FITC-labeled albumin concentration, and bradykinin exerted no marked difference.

Effects of NPY on intracellular cAMP content. Intracellular cAMP levels were obtained after administration of NPY in normoxia and hypoxia (Fig. 4). NPY concentration dependently diminished the intracellular cAMP contents significantly in normoxia (P < 0.05) and hypoxia (P < 0.001). At 10–7 and 3 x 10–7 M NPY, cAMP levels were significantly smaller in hypoxia than in normoxia (P < 0.05 and P < 0.01, respectively).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4. Effect of NPY on cAMP contents of RAECs. Intracellular cAMP was measured after cells were incubated in normoxia or hypoxia for 2 h in the presence of NPY. Values are means ± SE; n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 vs. 10–8 M NPY; P < 0.05; ¶¶P < 0.01 vs. normoxia.

 

Effects of NPY receptor agonists on RAEC monolayer permeability. To ascertain the effects of NPY receptor agonists on RAEC monolayer permeability, 10–6 M PYY, 10–5 M [Leu31,Pro34]-NPY-(13–36) (an NPY Y1 receptor agonist), or 10–5 M NPY-(13–36) (an NPY Y2 receptor agonist) was applied to RAEC monolayers 2 h after induction of hypoxia. FITC-labeled albumin concentration was 569.2 ± 93.6, 647.6 ± 125.1, and 604.8 ± 72.5 mg/l for PYY, [Leu31,Pro34]-NPY-(13–36), and NPY-(13–36), respectively; these values are not different from the control value (749.4 ± 67.3 mg/l).

Effects of NPY-(18–36), BIBP-3226, and L-NMMA on the NPY-induced increase in RAEC monolayer permeability. RAEC monolayers were pretreated with 10–5 M NPY-(18–36) (an NPY Y3 receptor antagonist), 3 x 10–5 M BIBP-3226 (an NPY Y1 receptor antagonist), or 10–5 M L-NMMA 10 min before administration of 3 x 10–7 M NPY into the upper chamber 2 h after induction of hypoxia. NPY-(18–36) prevented the response to NPY (P < 0.01), whereas BIBP-3226 and L-NMMA exerted no inhibitory action on the NPY-induced increase in FITC-labeled albumin concentration (Fig. 5).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. Effects of NPY Y3 receptor antagonist [NPY-(18–36)], NPY Y1 receptor antagonist [BIBP-3226 (BIBP)], and nitric oxide synthase inhibitor [N-monomethyl-L-arginine (L-NMMA)] on NPY-induced increase in RAEC monolayer permeability in hypoxia. Absorbance of FITC-labeled albumin in the lower chamber medium was measured 2 h after addition of FITC-labeled albumin into the upper chamber in hypoxia (5% O2). Cells were pretreated with 10–5 M NPY-(18–36), 3 x 10–5 M BIBP-3226, or 10–5 M L-NMMA 10 min before treatment with 3 x 10–7 M NPY. Values are means ± SE; n = 4. **P < 0.01 vs. no drug; ¶¶P < 0.01 vs. NPY.

 

Effects of FK-3657 or indomethacin on the NPY-induced increase in RAEC monolayer permeability. RAEC monolayers were pretreated with 10–5 M FK-3657 (a bradykinin B2 receptor antagonist) 10 min before administration of 3 x 10–7 M NPY into the upper chamber 2 h after induction of hypoxia. FK-3657 exerted no inhibitory action on the NPY-induced increase in FITC-labeled albumin concentration but blocked the bradykinin-induced increase in FITC-labeled albumin concentration (P < 0.01; Fig. 6). Pretreatment with 10–6 M indomethacin elicited no inhibitory action on the NPY-induced increase in albumin concentration in the lower chamber.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6. Effects of FK-3657 (FK) and indomethacin (Indo) on NPY- or bradykinin-induced increase in RAEC monolayer permeability in hypoxia. Absorbance of FITC-labeled albumin in the lower chamber medium was measured 2 h after addition of FITC-labeled albumin into the upper chamber in hypoxia. Cells were pretreated with 10–5 M FK-3657, a bradykinin B2 receptor antagonist, 10 min before treatment with 3 x 10–7 M NPY or 10–5 M bradykinin. Indomethacin, a cyclooxygenase inhibitor, was administered at 10–6 M 10 min before treatment with 3 x 10–7 M NPY. Values are means ± SE; n = 4. **P < 0.01 vs. no drug; ¶¶P < 0.01 vs. bradykinin.

 

Effects of tyrphostin SU-1498 on the NPY-induced increase in RAEC monolayer permeability. RAEC monolayers were pretreated with 10–6 M tyrphostin SU-1498 (a VEGF receptor-coupled tyrosine kinase inhibitor) 10 min before administration of 3 x 10–7 M NPY into the upper chamber 2 h after induction of hypoxia. SU-1498 exerted no inhibitory action on the NPY-induced increase in FITC-labeled albumin concentration but blocked the VEGF-induced increase in FITC-labeled albumin concentration (P < 0.01; Fig. 7).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7. Effects of tyrphostin SU-1498 (SU) on NPY- or vascular endothelial growth factor (VEGF)-induced increase in RAEC monolayer permeability with hypoxia. Absorbance of FITC-labeled albumin in the lower chamber medium was measured 2 h after addition of FITC-labeled albumin into the upper chamber in hypoxia. Cells were pretreated with 10–6 M SU-1498, a VEGF receptor-coupled tyrosine kinase inhibitor, 10 min before treatment with 3 x 10–7 M NPY or 10 ng/ml VEGF. Values are means ± SE; n = 4. **P < 0.01 vs. no drug; ¶¶P < 0.01 vs. VEGF.

 

Intracellular Ca2+ concentration after administration of NPY or PYY. Intracellular Ca2+ concentration before administration of NPY or PYY in hypoxia was 167.1 ± 34.0 nM. At 5 min after treatment with 3 x 10–7 M NPY and 10–6 M PYY, Ca2+ concentration was elevated to 420.9 ± 61.9 and 452.3 ± 101.5 nM, respectively (Fig. 8). These values were significantly greater than those obtained at 5 min without drugs (P < 0.01 for each). Within 2 h, the concentration tended to decrease toward the original level.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 8. Time trends of intracellular Ca2+ concentration after treatment with NPY or peptide YY (PYY) in hypoxia. Intracellular Ca2+ concentrations are plotted vs. time after injection of 3 x 10–7 NPY or 10–6 M PYY into the chamber medium in hypoxia. {square}, Data obtained from all groups for NPY, PYY, and no drug. Values are means ± SE; n = 4. **P < 0.01 vs. no drug.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results obtained in the present study demonstrated that NPY concentration dependently increases large molecule permeability across an RAEC monolayer under conditions of hypoxia. The increase in RAEC monolayer permeability caused by NPY was eventually consistent with the results reported by Hirabayashi et al. (14), who studied the capillary filtration coefficient, presumably under hypoxic conditions. However, the permeability was not significantly increased under normoxic conditions, suggesting that the RAEC monolayer permeability response to NPY is closely related to O2 content. Noll et al. (32), who showed a decrease in permeability using coronary arterial endothelial cells, performed experiments under aerated (~20% O2) conditions. The discrepancy in the permeability responses to NPY may be partly explained in terms of O2 tension.

The concentration-dependent decline in intracellular cAMP in RAECs treated with NPY in hypoxia corresponded to the increase in permeability. It has been reported that cAMP modulates endothelial monolayer permeability. Most investigators (26) suggested that an increase in cAMP inhibits the increase in vascular permeability. However, Noll et al. (32), using rat coronary endothelial cells in normoxia, reported that NPY decreased cAMP content and monolayer permeability, suggesting that some microvascular endothelial cells, especially the coronary arterial cells, differ from cells from most other vascular beds in the relation between vascular permeability and intracellular cAMP content. Other investigators (18, 44) also demonstrated differences in responses to some agonists between micro- and macrovascular endothelial cells. Furthermore, in RAECs, the effect of NPY on cAMP content was much smaller in normoxia than in hypoxia. As shown previously (6), hypoxia may diminish the cAMP content in endothelial cells; therefore, it is likely that the increase in RAEC monolayer permeability caused by NPY may be enhanced in hypoxia by the decrease in cAMP content.

Because NPY is present in the sympathetic nerves, it may have multiple physiological actions in the brain and peripheral nervous tissues. Sakakibara et al. (35) showed that electrical sympathetic nerve stimulation increased vascular permeability in isolated rat lung perfusion preparations, suggesting that NPY might mediate the response. NPY could, in part, by increasing vascular permeability in hypoxia, contribute to development of neurogenic pulmonary edema. In addition, NPY has been shown to be released by nerve injury, stress, and exercise (19, 24, 45). Thoresen et al. (40) and Fletcher et al. (7) demonstrated an increase in NPY serum concentration in hypoxia. In their reports, NPY serum concentration was 10–11–10–10 M, which is much lower than the effective concentration range of NPY obtained in the present study. Such a discrepancy in NPY concentration may be due to a dilution effect of serum and barrier effects of the vascular wall. A possible role of NPY in sympathetic nerve-related diseases, e.g., cardiovascular diseases such as coronary diseases and hypertension, and obesity/diabetes (2), may result in an increase in vascular endothelial permeability.

The NPY family of peptides includes endogenous substances such as PYY and the pancreatic polypeptide (2), both of which are synthesized in endocrine cells. NPY is primarily produced in neurons, together with norepinephrine in postganglionic sympathetic perivascular and myocardial neurons, and plays a central role in regulation of cardiovascular function (10). In the present study, PYY had no effect on RAEC monolayer permeability in normoxia or hypoxia. Furthermore, NPY Y1 and Y2 receptor agonists and NPY Y1 receptor antagonist elicited no remarkable effects, whereas NPY-(18–36), a weak NPY Y3 receptor antagonist and Y1/Y2 receptor agonist (1), prevented the NPY-induced increase in RAEC monolayer permeability. These results suggest that NPY-induced hyperpermeability across the RAEC monolayer may be mediated by the NPY Y3 receptor, because it is not activated by PYY and blocked by NPY-(18–36). In addition to its localization in vascular endothelial cells, the NPY Y3 receptor has been identified in rat superior cervical ganglia sympathetic neurons, rat nucleus tractus solitarii, rat cardiac myocytes, and rat distal colon (1, 5, 8, 11, 31).

Several intracellular or intercellular substrates, such as Ca2+, NO, bradykinin, and VEGF, have been shown to affect vascular endothelial permeability. In the present study, however, neither L-NMMA, FK-3657, nor tyrphostin SU-1498 blocked the NPY-induced elevation of RAEC monolayer permeability. Therefore, the NPY-induced increase in RAEC monolayer permeability did not appear to be attributable to NO. If a twofold or greater increase in Ca2+ concentration in response to NPY (Fig. 8) activates endothelial NO synthase activity (22), the amount of NO released may not be enough to elevate monolayer permeability. Similarly, the lack of effect of tyrphostin SU-1498, which blocked the VEGF-induced increase in permeability, indicated no role for VEGF. In addition, bradykinin may not participate in the NPY-induced increase in RAEC monolayer permeability because of lack of influence with FK-3657, a bradykinin B2 receptor antagonist that was developed for blocking the nasal congestion and bronchosub-mucosal permeability edema induced by bradykinin in cases of allergic airway diseases (15). Even if bradykinin causes NO release from endothelial cells via the B2 receptor (3), bradykinin seemed not to mediate the permeating action of NPY. Furthermore, indomethacin elicited no apparent inhibitory action on the NPY-induced elevation of monolayer permeability, suggesting that prostaglandins, products of cyclooxygenase, may not be involved in the action of NPY.

Hypoxia increases endothelial cell monolayer permeability through VEGF and NO, but these effects do not appear until a long time after exposure to 5% O2 hypoxia. The amount of VEGF produced by HUVECs (29) began to increase from 24 h after incubation with 5% O2 hypoxia and leveled off at 48 h. In the present study, the NPY-induced increase in permeability was greater at 2 h than at 24 h after hypoxia. The effects of bradykinin and histamine were quite different: the increase in permeability was smaller at 2 h than at 24 h after hypoxia. Furthermore, tyrphostin SU-1498 had no apparent effect on the NPY-induced increase in RAEC monolayer permeability at 2 h (Fig. 7) and 24 h (unpublished observation). These results indicate that VEGF may not contribute to the NPY-induced elevation of permeability.

NPY and PYY elevated the intracellular Ca2+ concentration within 5 min, whereas monolayer permeability was increased by NPY, but not by PYY. Several studies demonstrated that endothelial cytosolic Ca2+ and endothelial protein kinase C (PKC) are important regulators of endothelial permeability (23). Hypoxia, reportedly, increases human microvascular endothelial permeability through elevation of the intracellular Ca2+ concentration, activating NO synthase (12, 16, 22, 30), cGMP-dependent protein kinase, and PKC, which may control cadherin-occludin-cytoskeleton binding (33). Recently, Namiki et al. (29) showed that 5% O2 increased VEGF concentration in the culture medium of HUVECs, and VEGF increases microvascular permeability by a signaling cascade involving an increase in intracellular Ca2+ concentration (33), NO synthesis, cGMP-dependent protein kinase/PKC activation (43), and activation of extracellular signal-regulated kinase (17). In this context, the results obtained in the present study suggest that the increased permeability with NPY may not simply be explained by enhancement of a hypoxia-induced mechanism, i.e., an increase in intracellular Ca2+ concentration.

In conclusion, NPY, a sympathetic neurotransmitter, evokes an increase in vascular permeability in hypoxia via the NPY Y3 receptor. Because L-NMMA, FK-3657, indomethacin, and tyrphostin SU-1498 failed to inhibit such an increase in permeability, NPY seemed to elicit a direct action on endothelial cells, providing a research technique for evaluating the physiological function of the NPY Y3 receptor and specific antagonists relevant to its cloning.


   ACKNOWLEDGMENTS
 
GRANTS

This study was supported, in part, by Japanese Ministry of Education, Science, Sports, Culture, and Technology, Japan, Grants 09470325 and 12670097.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Ishikawa, Dept. of Pharmacology, Aichi Medical Univ., School of Medicine, Nagakute, Aichi 480-1195, Japan (E-mail: nao{at}aichi-med-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Balasubramaniam A, Sheriff S, Rigel DF, and Fischer JE. Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes. Peptides 11: 545–550, 1990.[CrossRef][Web of Science][Medline]
  2. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, and Bloom SR. Gut hormone PYY(3–36) physiologically inhibits food intake. Nature 418: 650–654, 2002.[CrossRef][Medline]
  3. Bergaya S, Meneton P, Bloch-Faure M, Mathieu E, Alhenc-Gelas F, Levy BI, and Boulanger CM. Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice. Circ Res 88: 593–599, 2001.[Abstract/Free Full Text]
  4. Chu SJ, Chang DM, Wang D, Chen YH, Hsu CW, and Hsu K. Fructose-1,6-diphosphate attenuates acute lung injury induced by ischemia-reperfusion in rats. Crit Care Med 30: 1605–1609, 2002.[CrossRef][Web of Science][Medline]
  5. Dumont Y, Satoh H, Cadieux A, Taoudi-Benchekroun M, Pheng LH, St.-Pierre S, Fournier A, and Quirion R. Evaluation of truncated neuropeptide Y analogues with modifications of the tyrosine residue in position 1 on Y1, Y2 and Y3 receptor sub-types. Eur J Pharmacol 238: 37–45, 1993.[CrossRef][Web of Science][Medline]
  6. Fischer S, Renz D, Wiesnet M, Schaper W, and Karliczek GF. Hypothermia abolishes hypoxia-induced hyperpermeability in brain microvessel endothelial cells. Brain Res Mol Brain Res 74: 135–144, 1999.[Medline]
  7. Fletcher AJW, Edwards CMB, Gardner DS, Fowden AL, and Giussani DA. Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation. Endocrinology 141: 3976–3982, 2000.[Abstract/Free Full Text]
  8. Foucart S, Bleakman D, Bindokas VP, and Miller RJ. Neuropeptide Y and pancreatic polypeptide reduce calcium currents in acutely dissociated neurons from adult rat superior cervical ganglia. J Pharmacol Exp Ther 265: 903–909, 1993.[Abstract/Free Full Text]
  9. Gray TS and Morley JE. Neuropeptide Y: anatomical distribution and possible function in the mammalian nervous system. Life Sci 38: 389–401, 1986.[CrossRef][Web of Science][Medline]
  10. Grundemar L and Hakanson R. Neuropeptide Y effector systems: perspective for drug development. Trends Pharmacol Sci 15: 153–159, 1994.[CrossRef][Medline]
  11. Grundemar L, Wahlestedt C, and Reis DJ. Neuropeptide Y acts at an atypical receptor to evoke cardiovascular depression and to inhibit glutamate responsiveness in the brainstem. J Pharmacol Exp Ther 258: 633–638, 1991.[Abstract/Free Full Text]
  12. Hampl V, Cornfield DN, Cowan NJ, and Archer SL. Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells. Eur Respir J 8: 515–522, 1995.[Abstract]
  13. Heilig M and Widerlov E. Neurobiology and clinical aspects of neuropeptide Y. Crit Rev Neurobiol 9: 115–136, 1995.[Web of Science][Medline]
  14. Hirabayashi A, Nishiwaki K, Shimada Y, and Ishikawa N. Role of neuropeptide Y and its receptor subtypes in neurogenic pulmonary edema. Eur J Pharmacol 296: 297–305, 1996.[CrossRef][Web of Science][Medline]
  15. Hirayama Y, Miyayasu K, Yamagami K, Imai T, Ohkubo Y, and Mutoh A. Effect of FK3657, a non-peptide bradykinin B2 receptor antagonist, on allergic airway disease models. Eur J Pharmacol 467: 197–203, 2003.[CrossRef][Web of Science][Medline]
  16. Huang QB and Yuan Y. Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability. Am J Physiol Heart Circ Physiol 273: H2442–H2451, 1997.[Abstract/Free Full Text]
  17. Kevil CG, Payne DK, Mire E, and Alexander JS. Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins. J Biol Chem 273: 15099–15103, 1998.[Abstract/Free Full Text]
  18. King GL, Buzney SM, Kahn CR, Hetu N, Buchwald S, Macdonald SG, and Rand LI. Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels. J Clin Invest 71: 974–979, 1983.[Web of Science][Medline]
  19. Landry M, Holmberg K, Zhang X, and Hokfelt T. Effect of axotomy on expression of NPY, galanin, and NPY Y1 and Y2 receptors in dorsal root ganglia and the superior cervical ganglion studied with double-labeling in situ hybridization and immunohistochemistry. Exp Neurol 162: 361–384, 2000.[CrossRef][Web of Science][Medline]
  20. Larhammar D. Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide. Regul Pept 65: 165–174, 1996.[CrossRef][Web of Science][Medline]
  21. Loesch A, Maynard KI, and Burnstock G. Calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in endothelial cells after long-term stimulation of perivascular nerves. Neuroscience 48: 723–726, 1992.[CrossRef][Web of Science][Medline]
  22. Luckhoff A, Pohl U, Mulsch A, and Busse R. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 95: 189–196, 1988.[Web of Science][Medline]
  23. Lum H and Malik AB. Regulation of vascular endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 267: L223–L241, 1994.[Abstract/Free Full Text]
  24. Lundberg JM, Martinsson A, Hemsen A, Theordorsson-Norheim E, Svedenhag J, Ekblom B, and Hjemdahl P. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem Biophys Res Commun 133: 30–36, 1985.[CrossRef][Web of Science][Medline]
  25. Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, and Westfall T. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 50: 143–150, 1998.[Abstract/Free Full Text]
  26. Minor T and Isselhard W. Cellular signal level of cyclic AMP and functional integrity of the small bowel after ischemic preservation: an experimental pilot study in the rat. Eur Surg Res 30: 144–148, 1998.[CrossRef][Web of Science][Medline]
  27. Mortelliti MP and Manning HL. Acute respiratory distress syndrome. Am Fam Physician 65: 1823–1830, 2002.[Web of Science][Medline]
  28. Myers AK, Farhat MY, Vaz CA, Keiser HR, and Zukowska-Grojec Z. Release of immunoreactive neuropeptide by rat platelets. Biochem Biophys Res Commun 155: 118–122, 1988.[CrossRef][Web of Science][Medline]
  29. Namiki A, Brogi E, Kearney M, Kim EA, Wu T, Couffinhal T, Varticovski L, and Isner JM. Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells. J Biol Chem 270: 31189–31195, 1995.[Abstract/Free Full Text]
  30. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051–3064, 1992.[Abstract]
  31. Norenberg W, Bek M, Limberger N, Takeda K, and Illes P. Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A system. Naunyn Schmiedebergs Arch Pharmacol 351: 337–347, 1995.[Web of Science][Medline]
  32. Noll T, Hempel A, and Piper HM. Neuropeptide Y reduces macromolecule permeability of coronary endothelial monolayers. Am J Physiol Heart Circ Physiol 271: H1878–H1883, 1996.[Abstract/Free Full Text]
  33. Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, and Alexander JS. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am J Physiol Cell Physiol 277: C1066–C1074, 1999.[Abstract/Free Full Text]
  34. Plateel M, Dehouck MP, Torpier G, Cecchelli R, and Teissier E. Hypoxia increases the susceptibility to oxidant stress and the permeability of the blood-brain barrier endothelial cell monolayer. J Neurochem 65: 2138–2145, 1995.[Web of Science][Medline]
  35. Sakakibara H, Hashiba Y, Taki K, Kawanishi M, Shimada Y, and Ishikawa N. Effects of sympathetic nerve stimulation on lung vascular permeability in the rat. Am Rev Respir Dis 145: 685–692, 1992.[Web of Science][Medline]
  36. Shine J, Potter EK, Biden T, Selbie LA, and Herzog H. Neuropeptide Y and regulation of the cardiovascular system. J Hypertens Suppl 12: S41–S45, 1994.[Medline]
  37. Snedecor GW and Cochran WG. Statistical Methods. Ames, IA: Iowa State University Press, 1967.
  38. Suh SH, Vennekens R, Manolopoulos VG, Freichel M, Schweig U, Prenen J, Flockerzi V, Droogmans G, and Nilius B. Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling. Pflügers Arch 438: 612–620, 1999.[CrossRef][Web of Science][Medline]
  39. Tatemoto K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA 79: 5485–5489, 1982.[Abstract/Free Full Text]
  40. Thoresen M, Dahlin I, Lundberg JM, and Lagercrantz H. Neuropeptide Y and catecholamine release in the piglet during hypoxia: enhancement by theophylline. J Dev Physiol 18: 187–191, 1992.[Web of Science][Medline]
  41. Wahlestedt C, Regunathan S, and Reis DJ. Identification of cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY. Life Sci 50: PL7–PL12, 1992.[CrossRef][Web of Science][Medline]
  42. Woldbye DP, Larsen PJ, Mikkelsen JD, Klemp K, Madsen TM, and Bolwig TG. Powerful inhibition of kainic acid seizures by neuropeptide Y via Y5-like receptors. Nat Med 3: 761–764, 1996.[Web of Science]
  43. Wu HM, Huang Q, Yuan Y, and Granger HJ. VEGF induces NO-dependent hyperpermeability in coronary venules. Am J Physiol Heart Circ Physiol 271: H2735–H2739, 1996.[Abstract/Free Full Text]
  44. Zink S, Rosen P, and Lemoine H. Micro- and macrovascular endothelial cells in {beta}-adrenergic regulation of transendothelial permeability. Am J Physiol Cell Physiol 269: C1209–C1218, 1995.[Abstract/Free Full Text]
  45. Zukowska-Grojec Z, Dayao EK, Karwatowska-Prokopczuk E, Hauser GJ, and Doods HN. Stress-induced mesenteric vasoconstriction in rats is mediated by neuropeptide Y Y1 receptors. Am J Physiol Heart Circ Physiol 270: H796–H800, 1996.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Z.-Y. Chen, G.-G. Feng, K. Nishiwaki, Y. Shimada, Y. Fujiwara, T. Komatsu, and N. Ishikawa
Possible roles of neuropeptide Y Y3-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction
Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H959 - H967.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
N. Kurimoto, Y.-S. Nan, Z.-Y. Chen, G.-G. Feng, T. Komatsu, N. Kandatsu, J. Ko, N. Kawai, and N. Ishikawa
Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF
Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H100 - H106.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/3/H1027    most recent
00630.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nan, Y.-S.
Right arrow Articles by Ishikawa, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nan, Y.-S.
Right arrow Articles by Ishikawa, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.