Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 93: 1377-1383, 2002; doi:10.1152/japplphysiol.00277.2002
8750-7587/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI 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 ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsueshita, T.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsueshita, T.
Right arrow Articles by Rubinstein, I.
Vol. 93, Issue 4, 1377-1383, October 2002

Phospholipids modulate the biophysical properties and vasoactivity of PACAP-(138)

Takaya Tsueshita1,3, Salil Gandhi2, Hayat Önyüksel1,2, and Israel Rubinstein1,3,4

Departments of 1 Biopharmaceutical Sciences, 2 Bioengineering, and 3 Medicine, University of Illinois at Chicago, and 4 Chicago Veterans Afffairs Health Care System, West Side Division, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to elucidate the interactions between pituitary adenylate cyclase-activating peptide (PACAP)-(138) and phospholipids in vitro and to determine whether these phenomena modulate, in part, the vasorelaxant effects of the peptide in the intact peripheral microcirculation. We found that the critical micellar concentration of PACAP-(138) was 0.4-0.9 µM. PACAP-(138) significantly increased the surface tension of a dipalmitoylphosphatidylcholine monolayer and underwent conformational transition from predominantly random coil in saline to alpha -helix in the presence of distearoyl-phosphatidylethanolamine-polyethylene glycol (molecular mass of 2,000 Da) sterically stabilized phospholipid micelles (SSM) (P < 0.05). Using intravital microscopy, we found that aqueous PACAP-(138) evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch that was significantly potentiated when PACAP-(138) was associated with SSM (P < 0.05). The vasorelaxant effects of aqueous PACAP-(138) were mediated predominantly by PACAP type 1 (PAC1) receptors, whereas those of PACAP-(138) in SSM predominantly by PACAP/vasoactive intestinal peptide type 1 and 2 (VPAC1/VPAC2) receptors. Collectively, these data indicate that PACAP-(138) self-associates and interacts avidly with phospholipids in vitro and that these phenomena amplify peptide vasoactivity in the intact peripheral microcirculation.

neuropeptide; amphipathic; micelles; distearoyl-phosphatidylethanolamine-polyethylene glycol; vasodilation; receptor antagonist; hamster


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PITUITARY ADENYLATE CYCLASE-activating peptide (PACAP)-(138) is a 38-amino acid pleiotropic neuropeptide originally isolated from the ovine hypothalamus (2). It is a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily of peptides and shares 68% sequence homology with VIP (2, 43). It is well established that the peptide is widely distributed in the peripheral microcirculation and on release elicits potent vasodilation (2, 13, 27, 42, 43). However, this response is short lived, most likely because of proteolytic inactivation of the PACAP-(138) (13, 27, 43). Whether the vasorelaxant effects of PACAP-(138) are amplified by intervention that promote peptide stability is uncertain.

To this end, previous work from our laboratory showed that VIP self-assembles in an aqueous solution to form micelles, interacts avidly with a biomimetic membrane to increase its surface pressure, and undergoes conformational transition from predominantly random coil in aqueous solutions to alpha -helix in the presence of sterically stabilized phospholipid micelles (SSM) (29, 35). Self-association of VIP with SSM evoked significant potentiation and prolongation of the vasorelaxant effects of the peptide in the intact peripheral microcirculation relative to aqueous VIP (11, 20, 30, 35-40). Whether PACAP-(138) expresses similar properties is uncertain.

Hence, the purpose of this study was to begin to address these issues by determining whether PACAP-(138) self-assembles in an aqueous environment, increases surface pressure of a biomimetic phospholipid monolayer, and undergoes conformational transition in phospholipids and, if so, whether these phenomena potentiate and prolong PACAP-(138)-induced vasodilation in the intact peripheral microcirculation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular Interactions of PACAP-(138) In Vitro

Critical micellar concentration of PACAP-(138). First, the surface tension of HEPES buffer (pH 7.4) contained in a 30-ml custom-made Teflon trough was determined at room temperature (25°C) by using a DuNouy surface tensiometer (model 21 Tensiomat, Fisher Scientific, Pittsburgh, PA) (10, 29). Then, 50 µl of increasing concentrations of PACAP-(138) (0.1-1.0 µM final concentrations in the trough) dissolved in HEPES buffer (pH 7.4) were injected into the subphase through a side port and the surface tension of the PACAP-(138) solution was determined. The concentration above which surface tension no longer significantly decreased was considered as the critical micellar concentration (CMC) of PACAP-(138) (10, 29, 45).

Effects of PACAP-(138) on surface pressure of a phospholipid monolayer. Dipalmitoylphosphatidylcholine (DPPC; 10 µM) was dissolved in hexane-ethanol (9:1 vol/vol), and 50 µl of the solution were spread over 28 ml of HEPES buffer contained in a cylindrical 30-ml custom-made Teflon trough (internal diameter, 9 cm) at room temperature (25°C). A DPPC monolayer is formed spontaneously at the buffer-air interface after total evaporation of the organic solvent in 5 min. Surface tension of the monolayer was determined every 5 min for 60 min and every 10 min for 60 min thereafter by using a DuNouy surface tensiometer. Surface pressure was defined as the difference in surface tension between the DPPC monolayer and HEPES buffer alone as previously described in our laboratory (10, 29, 45). PACAP-(138) (1.5 and 3.6 µM final concentration in the trough) was injected into the subphase through a side port without disturbing the DPPC monolayer. Surface tension of the DPPC monolayer containing PACAP-(138) was then determined as outlined above.

Conformation of PACAP-(138). PACAP-(138) self-associated with SSM composed of distearoyl-phosphatidylethanolamine polyethylene glycol (molecular mass of 2,000 Da) (DSPE-PEG 2000) was prepared by using a method previously described in our laboratory (10, 35). Briefly, DSPE-PEG 2000 (1.0 mM) was dissolved in saline and mixed to form SSM. The resulting suspension was then incubated with PACAP-(138) for 2 h at room temperature (25°C) before use. The size of SSM alone and of PACAP-(138) in SSM was 18 ± 1 nm as determined by quasi-elastic light scattering (QELS; model 380, Nicomp Submicron Particle Sizer, Pacific Scientific, Menlo Park, CA).

The secondary structure of PACAP-(138) was determined by circular dichroism. Spectra were recorded on a spectropolarimeter (model J-700, JASCO, Easton, MD) at room temperature (25°C) by using a fused quartz cell of 1-cm pathlength containing saline with PACAP-(138) (4 µM) alone and PACAP-(138) (4 µM) incubated with DSPE-PEG 2000 (1 mM) for 2 h. A bandwidth of 1.0 nm and a step resolution of 0.5 nm were used to collect an average of five accumulations per sample at the near-ultraviolet range (190- to 260-nm wavelength) (9, 10, 16, 24, 29, 35). The acquired spectra were corrected to the baseline by using saline and empty SSM and were averaged. The peptide spectra were smoothed by using the noise reduction function. Data are expressed as percentage of alpha -helix by using the equation %helicity = [-(theta  + 4,000)/29,000] × 100 (where theta  is ellipiticty) and were calculated by Selcon, Softsec version 1.2 (Softwood, Brookfield, CT) (10, 16, 35). The concentrations of aqueous PACAP-(138) and PACAP-(138) in SSM used in these experiments are based on previous studies with VIP and secretin in our laboratory (6, 10, 11, 20, 29, 30, 35-40).

Effects of PACAP-(138) on Arteriolar Diameter In Vivo

Preparation of animals. Adult golden Syrian hamsters (120-140 g body wt) were anesthetized with pentobarbital sodium (6 mg/100 g body wt ip). A tracheostomy was performed to facilitate spontaneous breathing. The left femoral vein was cannulated to inject supplemental anesthesia (2-4 mg · 100 g body wt-1 · h-1) during the experiment. A catheter was inserted into the left femoral artery and connected to a computer-monitored pressure transducer to record systemic arterial pressure and heart rate (Workbench for Windows+, Kent Scientific, Torrington, CT), which did not change significantly during the experiment. Body temperature was monitored and maintained constant (37-38°C) throughout the experiment by using a heating pad.

To visualize the microcirculation of the cheek pouch, we used an established method in our laboratory and the literature (7, 10-12, 20, 30, 31, 34-40). Briefly, the left cheek pouch was spread over the small plastic baseplate, and an incision was made in the outer skin to expose the cheek pouch membrane. The avascular connective tissue layer of the membrane was removed, and a plastic chamber was positioned over the baseplate and secured in place by suturing the skin around the upper chamber. This chamber was connected to a reservoir containing warmed bicarbonate buffer (37-38°C), which allowed continuous suffusion onto the cheek pouch. The bicarbonate buffer was bubbled continuously with 95% N2-5% CO2 (pH 7.4). The chamber was also connected via a three-way valve to an infusion pump (Sage Instruments, Boston, MA) that allowed constant administration of drugs into the suffusate.

Determination of arteriolar diameter. The cheek pouch microcirculation was visualized with a microscope (Nikon, Tokyo, Japan) coupled to a 100-W mercury light source at a final magnification of ×40. The microscope image was projected through a low-light television camera (Panasonic TR-124 MA, Matsushita Communication Industrial, Yokohama, Japan) onto a video screen (Panasonic). The inner diameter of second-order arterioles (baseline diameter, 48-60 µm), which modulate microvascular resistance in the cheek pouch (7, 31), was determined during the experiment from the video display of the microscope image by using a videomicrometer (model VIA 100, Boeckler Instruments, Tuscon, AZ; resolution, ±1 µm). In each animal, the same arteriolar segment was used to measure vessel diameter during the experiment.

Experimental Design

PACAP-(138)-induced vasodilation. The purpose of these studies was to determine whether self-association of PACAP-(138) with SSM potentiates and prolongs the vasorelaxant effects of the peptide in the intact cheek pouch microcirculation. Animals were divided into six groups, with four animals in each group. After suffusion of the bicarbonate buffer for 30 min (equilibration period), the cheek pouch was suffused with increasing concentrations of PACAP-(138) (0.01, 0.1, and 1.0 nmol) in saline or in SSM. Each concentration was suffused for 7 min in an arbitrary fashion. At least 45 min elapsed between subsequent suffusions of PACAP-(138) (30, 35, 37). Arteriolar diameter was determined every 5 min during the equilibration period, immediately before, every minute during, and after suffusion of PACAP-(138) until arteriolar diameter returned to baseline. In preliminary studies, we determined that repeated suffusions of PACAP-(138) (0.01, 0.1, and 1.0 nmol) in saline and in SSM were associated with reproducible results (each group, n = 4 animals; P > 0.5; data not shown). In addition, suffusions of saline [vehicle of PACAP-(138) and PACAP-(138) in SSM] and of empty SSM for the duration of the experiments were associated with no significant changes in arteriolar diameter (each group, n = 4 animals; P > 0.5; data not shown). The concentrations of PACAP-(138) in saline and in SSM used in these experiments were based on preliminary studies.

Effects of receptor antagonists on PACAP-(138)-induced vasodilation. The purpose of these studies was to determine whether the vasorelaxant effects of PACAP-(138) in the intact cheek pouch are mediated by activation of specific receptors. Four groups (each, n = 4 animals) were studied. After the equilibration period, the cheek pouch was suffused for 7 min with PACAP-(138) in saline (1.0 nmol) or PACAP-(138) in SSM (0.1 nmol). These concentrations were chosen because they elicited vasodilation of similar magnitude in the experiments outlined above. Thereafter, bicarbonate buffer was suffused for 30 min followed by 30-min suffusion of PACAP-(638) (10 nmol), a PACAP-(138) receptor antagonist (8, 17, 18, 22, 25, 26, 32, 41), or VIP-(1028) (10 nmol), a VIP-receptor antagonist (1, 5, 8, 16, 23, 37), followed by 7-min suffusion of PACAP-(138) in saline (1.0 nmol) or PACAP-(138) in SSM (0.1 nmol). Arteriolar diameter was determined after each intervention as outlined above. In preliminary experiments, we determined that 30-min suffusion of PACAP-(638) and VIP-(1028) (each, 10 nmol) alone had no significant effects on arteriolar diameter (each group, n = 4 animals; P > 0.5; data not shown). The concentrations of PACAP-(638) and VIP-(1028) used in these experiments are based on preliminary studies and previous reports from our laboratory and in the literature (21, 27, 37).

Chemicals and Drugs

Human PACAP-(138) was synthesized by Dr. Robert Lee at the Protein Research Laboratory, University of Illinois at Chicago (Chicago, IL). VIP-(1028) and PACAP-(638) were obtained from American Peptide (Sunnyvale, CA). Bicarbonate buffer (in mM: 131.9 NaCl, 0.76 MgCl2, 2.95 KCl, 11.87 NaHCO3, and 1.48 CaCl2) and HEPES were obtained from Sigma Chemical (St. Louis, MO). DSPE-PEG 2000 and DPPC were obtained from Avanti Polar Lipids (Alabaster, AL). All drugs were prepared and diluted in saline to the desired concentrations on the day of the experiment.

Data and Statistical Analyses

When a compound was suffused onto the cheek pouch, we determined the maximal change in arteriolar diameter and considered it the response to that compound in each animal. Arteriolar diameter was expressed as the ratio of experimental diameter to control diameter, with control diameter normalized to 100%, to account for intra-animal and interanimal variability. Data are expressed as means ± SE except for the size of micelles, which is expressed as means ± SD because these data were not used to compare between experimental groups. Statistical analysis was performed by using repeated-measures analysis of variance with Neuman-Keuls multiple-range post hoc test to detect values that were different from control values. A value of P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular interactions of PACAP-(138) In Vitro

CMC of PACAP-(138). The effects of injection of PACAP-(138) into the subphase of HEPES buffer (pH 7.4) on surface tension are shown in Fig. 1. The peptide elicited a significant concentration-dependent decrease in surface tension from baseline (Fig. 1; n = 3 experiments; P < 0.05). No significant change was observed above PACAP-(138) concentration of 0.4-0.9 µM, so this range was taken as the CMC of PACAP-(138) (Fig. 1). The size of PACAP-(138) micelles was below the lower detection limit of the QELS apparatus (5 nm).


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1.   Critical micellar concentration of pituitary adenylate cyclase-activating peptide (PACAP)-(138). Values are means ± SE; n = 3 experiments. *P < 0.05 compared with baseline.

Effects of PACAP-(138) on membrane surface pressure of a phospholipid monolayer. The effects of PACAP-(138) on surface pressure of a DPPC monolayer is depicted in Fig. 2. The DPPC monolayer alone is unstable at the air-water interface and decomposes shortly after spreading, leading to a sustained decline in surface pressure relative to baseline (Fig. 2; n = 3 animals; P < 0.05). By contrast, injection of PACAP-(138) (1.5 and 3.6 µM) into the subphase stabilized the DPPC monolayer as manifested by a dose-dependant increase in surface pressure of the monolayer over a sustained period of time (Fig. 2; each group, n = 3 experiments; P < 0.05). The increase in the surface pressure during the first 20 min of the experiment indicates incorporation of PACAP-(138) into the monolayer (Fig. 2; each group, n = 3 experiments; P < 0.05).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2.   Effects of PACAP-(138) (1.5 and 3.6 µM) on surface pressure of a dipalmitoylphosphatidylcholine (DPPC; 10 µM) monolayer. Values are means ± SE; each group, n = 3 experiments. *P < 0.05 compared with DPPC alone.

Conformation of PACAP-(138). The conformation of PACAP-(138) in saline at room temperature was unordered (alpha -helix content, 3.0 ± 1.0%; n = 3 experiments). By contrast, association of PACAP-(138) with DSPE-PEG 2000 at room temperature was associated with a significant increase in the proportion of alpha -helix [alpha -helix content, 13.0 ± 4.1%; n = 3 experiments; P < 0.05 compared with PACAP-(138) in saline].

Effects of PACAP-(138) on Arteriolar Diameter in Vivo

PACAP-(138)-induced vasodilation. Suffusion of PACAP-(138) elicited a significant, concentration-dependent increase in arteriolar diameter (Fig. 3; each group, n = 4 animals; P < 0.05 compared with baseline). Vasodilation was observed within 1-2 min after the start of suffusion, was maximal within 5-6 min, and returned to baseline within 25 min thereafter. Self-association of PACAP-(138) with SSM significantly potentiated the vasorelaxant effects of the peptide (Fig. 3; each group, n = 4 animals; P < 0.05 compared with PACAP in saline). In addition, the duration of vasodilation evoked by PACAP-(138) in SSM was significantly longer than that elicited by PACAP-(1-38) in saline (Fig. 4, each group, n = 4 animals; P < 0.05).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of PACAP-(138) in saline and PACAP-(138) in sterically stabilized phospholipid micelles (SSM) on arteriolar diameter expressed as percent change from baseline in the intact hamster cheek pouch. Values are means ± SE; each group, n = 4 animals. *P < 0.05 compared with baseline. +P < 0.05 compared with PACAP-(138) in saline.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Duration of vasodilation evoked by PACAP-(138) in saline and PACAP-(138) in SSM in the intact hamster cheek pouch. Values are means ± SE; each group, n = 4 animals. *P < 0.05 compared with baseline. +P < 0.05 compared with PACAP-(138) in saline.

Effects of receptor antagonists on PACAP-(138)-induced vasodilation. Suffusion of PACAP-(638) (10 nmol) significantly, and to a similar extent, attenuated the magnitude and duration of vasodilation elicited by both PACAP-(138) in saline (1.0 nmol) and PACAP in SSM (0.1 nmol) [Figs. 5 and 6; each group, n = 4 animals; P < 0.05 compared with PACAP-(138) in saline and PACAP-(138) in SSM alone]. Suffusion of VIP-(1028) (10 nmol) significantly attenuated the magnitude and duration of vasodilation elicited by PACAP-(138) in SSM (0.1 nmol) but not by PACAP-(138) in saline (1.0 nmol) [Figs. 5 and 6; each group, n = 4 animals; P < 0.05 compared with PACAP-(638) in SSM alone]. Concentrations of VIP-(1028) and PACAP-(638) higher than 10 nmol were not used in these experiments because they display agonist-like activity in the cheek pouch (37).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of PACAP-(138) in saline and PACAP-(138) in SSM on arteriolar diameter expressed as percent change from baseline in the absence and presence of vasoactive intestinal peptide (VIP)-(1028) (10 nmol) or PACAP-(638) (10 nmol) in the intact hamster cheek pouch. Values are means ± SE; each group, n = 4 animals. *P < 0.05 compared with baseline. +P < 0.05 compared with PACAP-(138) suffused with VIP-(1028) or PACAP-(638).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 6.   Duration of vasodilation evoked by PACAP-(138) in saline and PACAP-(138) in SSM in the absence and presence of VIP-(1028) (10 nmol) or PACAP-(638) (10 nmol) in the intact hamster cheek pouch. Values are means ±SE; each group, n = 4 animals. *P < 0.05 compared with baseline. +P < 0.05 compared with PACAP-(138) in saline.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are three new findings of this study. First, we found that PACAP-(138), a pleiotropic neuropeptide with a calculated net charge of +7 (2, 43), self-assembles in an aqueous solution in vitro with a CMC of 0.4-0.9 µM. Second, PACAP-(138), at concentrations detected in the synaptic cleft (2, 8, 43), increases significantly the surface pressure of a biomimetic monolayer over a sustained period of time. Taken together, these data indicate that PACAP-(138) at physiological concentrations is an amphipathic neuropeptide because it self-aggregates in an aqueous environment and interacts avidly with phospholipids.

This conclusion is supported by the observed change in molecular conformation of PACAP-(138) in the presence of DSPE-PEG 2000. There was a significant transition of the secondary structure of PACAP-(138) from predominantly random coil in saline to alpha -helix in the presence of phospholipids. alpha -Helix is the optimal conformation for PACAP-(138) to interact with its receptors in target cells and also confers stability to the peptide (3, 9, 10, 14, 16, 24, 28, 29, 33, 35, 44). To this end, Gao et al. (11) showed that association of VIP with liposomes protects the peptide from degradation by trypsin in vitro. Whether self-association of PACAP-(138) with SSM also protects the peptide from proteolysis remains to be determined.

The third new finding of this study is that suffusion of aqueous (random coil) PACAP-(138) onto the intact hamster cheek pouch microcirculation elicited significant concentration-dependent vasodilation that was attenuated by PACAP-(638), a PACAP type 1 (PAC1) and PACAP/VIP type 2 (VPAC2) receptor antagonist, but not by VIP-(1028), a PACAP/VIP type 1 (VPAC1)/VPAC2 receptor antagonist (1, 5, 17, 18, 22, 23, 26, 32, 41-43). Self-association of PACAP-(138) with SSM [alpha -helix PACAP-(138)] significantly potentiated and prolonged vasodilation evoked by the peptide relative to that elicited by aqueous PACAP-(138). However, unlike aqueous PACAP-(138), the vasorelaxant effects of PACAP-(138) in SSM were attenuated by both VIP-(1028) and PACAP-(638). The magnitude of micellar PACAP-(138)-induced vasodilation and its duration were similar to those evoked by micellar VIP in the cheek pouch (30, 35). Collectively, these data suggest that the spontaneous interactions of amphipathic neuropeptides with SSM in vitro are driven by common biophysical properties.

The mechanisms underlying PACAP-(138) interactions with phospholipids were not elucidated in this study. Nonetheless, the results of this study suggest that PACAP-(138) is attracted to and inserts itself into a phospholipid monolayer and SSM. This process is governed, most likely, by electrostatic and hydrophobic forces, leading to an increase in the membrane surface pressure (10, 29, 45). Although the physiological significance of these phenomena are uncertain, they nonetheless suggest that PACAP-(138) interacts directly with phospholipids in the plasma and organelle membranes in target cells, thereby altering membrane fluidity and intracellular signal transduction pathway(s) (10, 14, 28-30). Whether amphipathic neuropeptides, such as PACAP-(138), VIP, and secretin, associate spontaneously with phospholipids, such as lipoproteins, to form micelles in biological fluids remains to be established.

The hamster cheek pouch is an established animal model to elucidate the mechanisms underlying the vasoactive effects of various endogenous mediators, including VIP and PACAP-(138), in the intact peripheral microcirculation (7, 11, 12, 30, 31, 34-40). This intravital preparation is stable for at least 6 h, thereby allowing each animal to be used as its own control. This, in turn, reduces the number of animals required for each experiment and simplifies data analysis (11, 12, 30, 34-40).

The results of this study indicate that phospholipid-induced transition of PACAP-(138) secondary structure from random coil to alpha -helix not only amplifies vasodilation evoked by the peptide in the intact peripheral microcirculation but also shifts the receptor subtype activated by PACAP-(138) from predominantly PAC1 receptors for aqueous (random coil) PACAP-(138) to predominantly VPAC1/VPAC2 receptors for phospholipid-associated (alpha -helix) PACAP-(138). These data suggest a promiscuous behavior of PACAP-(138) in target cells, which is dependent, in part, on the biophysical state of the peptide and phospholipids in the interstitial fluid-plasma membrane interface (15, 32, 43). Clearly, the molecular mechanisms and intracellular signal transduction pathway(s) underlying this proposed phospholipid-dependent receptor shift in the intact peripheral microcirculation and its relevance to other biological effects of PACAP-(138) in vivo should be further probed by using more selective PACAP-(138) agonists and PAC1, VPAC1, and VPAC2 receptor antagonists as they become commercially available (43).

In summary, we found that PACAP-(138) is an amphipathic neuropeptide that self-assembles in an aqueous solution and interacts avidly with a biomimetic monolayer, leading to a significant and sustained increase in the surface pressure of the monolayer. In addition, PACAP-(138) undergoes conformational transition from predominantly random coil in saline to alpha -helix in the presence of SSM. Importantly, self-association of PACAP-(138) with sterically stabilized phospholipid micelles potentiates and prolongs the vasorelaxant effects of the peptide in the intact peripheral microcirculation while shifting its receptor subtype specificity from predominantly PAC1 receptors to predominantly VPAC1/VPAC2 receptors. We suggest that PACAP-(138) interactions with phospholipids modulate, in part, its vasoactive effects in vivo.


    ACKNOWLEDGEMENTS

This study was supported, in part, by the Campus Research Board, University of Illinois at Chicago.


    FOOTNOTES

Address for reprint requests and other correspondence: I. Rubinstein, Dept. of Biopharmaceutical Sciences (M/C 865), Univ. of Illinois at Chicago, 833 South Wood St., Chicago, IL 60612-7231 (E-mail: IRubinst{at}uic.edu).

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.

10.1152/japplphysiol.00277.2002

Received 1 April 2002; accepted in final form 11 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adamou, JE, Aiyar N, Van Horn S, and Elshourbagy NA. Cloning and functional characterization of the human vasoactive intestinal peptide (VIP)-2 receptor. Biochem Biophys Res Commun 209: 385-392, 1995[ISI][Medline].

2.   Arimura, A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 37: 287-303, 1992[ISI][Medline].

3.   Bodanszky, M, Bodanszky A, Klausner YS, and Said SI. A preferred conformation in the vasoactive intestinal peptide (VIP). Molecular architecture of gastrointestinal hormones. Bioorg Chem 3: 133-140, 1974.

4.   Brizzolara, AL, and Burnstock G. Endothelium-dependent and endothelium-independent vasodilatation of the hepatic artery of the rabbit. Br J Pharmacol 103: 1206-1212, 1991[ISI][Medline].

5.   Couvineau, A, Rouyer-Fessard C, Darmoul D, Maoret JJ, Carrero I, Ogier-Denis E, and Laburthe M. Human intestinal VIP receptor: cloning and functional expression of two cDNA encoding proteins with different N-terminal domains. Biochem Biophys Res Commun 200: 769-776, 1994[ISI][Medline].

6.   Dagar, S, Sekosan M, Rubinstein I, and Önyüksel H. Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting. Breast Cancer Res Treat 65: 49-54, 2001[ISI][Medline].

7.   Davis, MJ, Joyner WL, and Gilmore JP. Microvascular pressure distribution and responses of pulmonary allografts and cheek pouch arterioles in the hamster to oxygen. Circ Res 49: 125-132, 1981[Abstract/Free Full Text].

8.   Dickinson, T, Fleetwood-Walker SM, Mitchell R, and Lutz EM. Evidence for roles of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) receptors in modulating the responses of rat dorsal horn neurons to sensory inputs. Neuropeptides 31: 175-185, 1997[ISI][Medline].

9.   Filizola, M, Carteni-Farina M, and Perez JJ. Conformational study of vasoactive intestinal peptide by computational methods. J Pept Res 50: 55-64, 1997[ISI][Medline].

10.   Gandhi, S, Rubinstein I, Tsueshita T, and Önyüksel H. Secretin self-assembles and interacts spontaneously with phospholipids in vitro. Peptides 23: 201-204, 2002[ISI][Medline].

11.   Gao, XP, Noda Y, Rubinstein I, and Paul S. Vasoactive intestinal peptide encapsulated in liposomes: effects on systemic arterial blood pressure. Life Sci 54: PL247-PL252, 1994[ISI][Medline].

12.   Gao, XP, Suzuki H, Olopade CO, and Rubinstein I. Short-term exposure to lipopolysaccharide is associated with microvascular contractile dysfunction in vivo. Life Sci 56: 1243-1249, 1995[ISI][Medline].

13.   Gardiner, SM, Rakhit T, Kemps PA, March JE, and Bennett T. Regional haemodynamic responses to PACAP and VIP in conscious rats. Br J Pharmacol 111: 589-597, 1994[ISI][Medline].

14.   Gololobov, G, Noda Y, Sherman S, Rubinstein I, Baranowska-Kortylewicz J, and Paul S. Stabilization of vasoactive intestinal peptide by lipids. J Pharmacol Exp Ther 285: 753-758, 1998[Abstract/Free Full Text].

15.   Greaves, RRSH, O'Donnell LJD, Battistini B, Forget MA, and Farthing MJG The differential effect of VIP and PACAP on guinea pig gallbladder in vitro. Eur J Gastroenterol Hepatol 12: 1181-1184, 2000[ISI][Medline].

16.   Haghjoo, K, Cash PW, Farid RS, Komisaruk BR, Jordan F, and Pochapsky SS. Solution structure of vasoactive intestinal peptide (1128)-NH2, a fragment with analgesic properties. Pept Res 9: 327-331, 1996[ISI][Medline].

17.   Hashimoto, H, Ishihara T, Shigemoto R, Mori K, and Nagata S. Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase activating polypeptide. Neuron 11: 333-342, 1993[ISI][Medline].

18.   Hosoya, M, Onda H, Ogi K, Masuda Y, Miyamoto Y, Ohtaki T, Okazaki H, Arimura A, and Fujino M. Molecular cloning and functional expression of rat cDNAs encoding the receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Biochem Biophys Res Commun 194: 133-143, 1993[ISI][Medline].

19.   Ignarro, LJ, Byrns RE, Buga GM, and Wood KS. Mechanisms of endothelium-dependent vascular smooth muscle relaxation elicited by bradykinin and VIP. Am J Physiol Heart Circ Physiol 253: H1074-H1082, 1987[Abstract/Free Full Text].

20.   Ikezaki, H, Paul S, Alkan-Onyuksel H, Patel M, Gao XP, and Rubinstein I. Vasodilation elicited by liposomal VIP is unimpeded by anti-VIP antibody in hamster cheek pouch. Am J Physiol Regul Integr Comp Physiol 275: R56-R62, 1998[Abstract/Free Full Text].

21.   Kobayashi, H, Uezono Y, Ueno S, and Izumi F. Pituitary adenylate cyclase activating polypeptides (PACAPs) increase cAMP in rat cerebral microvessels. Brain Res 647: 145-147, 1994[ISI][Medline].

22.   Lenard, L, Jr, Lazar Z, Benko R, Szigeti R, Bathori Z, Toth GK, Penke B, and Bartho L. Inhibitory effect of PACAP(638) on relaxations induced by PACAP, VIP and non-adrenergic, non-cholinergic nerve stimulation in the guinea-pig taenia caeci. Naunyn-Schmiedebergs Arch Pharmacol 361: 492-497, 2000[ISI][Medline].

23.   Lutz, EM, Sheward WJ, West KM, Morrow JA, Fink G, and Harmer AJ. The VIP2 receptor: molecular characterization of a cDNA encording a novel receptor for vasoactive intestinal peptide. FEBS Lett 334: 3-8, 1993[ISI][Medline].

24.   Lee, S, Iwata T, Oyagi H, Aoyagi H, Ohno M, Anaie K, Kirino Y, and Sugihara G. Effects of salts on conformational changes of basic amphipathic peptides from beta -structure to alpha -helix in the presence of phospholipid liposomes and their channel-forming ability. Biochim Biophys Acta 1151: 76-82, 1993[Medline].

25.   Leyton, J, Gozez Y, Pisegua J, Coy D, Purdom S, Casibang M, Zia F, and Moody TW. PACAP(638) is a PACAP receptor antagonist for breast cancer cells. Breast Cancer Res Treat 56: 177-186, 1999[ISI][Medline].

26.   Morrow, JA, Lutz EM, West KM, Fink G, and Harmar A. J Molecular cloning and expression of a cDNA encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). FEBS Lett 329: 99-105, 1993[ISI][Medline].

27.   Naruse, S, Suzuki T, Ozaki T, and Nokihara K. Vasodilator effect of pituitary adenylate cyclase activating polypeptide (PACAP) on femoral blood flow in dogs. Peptides 14: 505-510, 1993[ISI][Medline].

28.   Noda, Y, Rodriguez-Sierra J, Liu J, Landers D, Mori A, and Paul S. Partitioning of vasoactive intestinal polypeptide into lipid bilayers. Biochim Biophys Acta 1191: 324-330, 1994[Medline].

29.   Önyüksel, H, Bodalia B, Sethi V, Dagar S, and Rubinstein I. Surface-active properties of vasoactive intestinal peptide. Peptides 21: 419-423, 2000[ISI][Medline].

30.   Önyüksel, H, Ikezaki H, Patel M, Gao XP, and Rubinstein I. A novel formulation of VIP in sterically stabilized micelles amplifies vasodilation in vivo. Pharm Res 16: 155-160, 1999[ISI][Medline].

31.   Raud, J. Intravital microscopic studies on acute mast cell-dependent inflammation. Acta Physiol Scand Suppl 578: 1-58, 1989[Medline].

32.   Robberccht, P, Gourlet P, Neef DP, Woussen-Cole MC, Vandermeers-Piret MC, Vandermeers A, and Christophe J. Structural requirements for the occupancy of pituitary adenylate cyclase activating peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP(638) as a potent antagonist. Eur J Biochem 207: 239-246, 1992[ISI][Medline].

33.   Robinson, RM, Blakeney EW, Jr, and Mattice WL. Lipid-induced conformational changes in glucagons, secretin, and vasoactive intestinal peptide. Biopolymers 21: 1217-1228, 1982.

34.   Rubinstein, I, and Mayhan WG. L-Arginine dilates cheek pouch arterioles in hamsters with hereditary cardiomyopathy. J Lab Clin Med 125: 313-318, 1995[ISI][Medline].

35.   Rubinstein, I, Patel M, Ikezaki H, Dagar S, and Önyüksel H. Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin. Peptides 20: 1497-1501, 2000.

36.   Séjourné, F, Rubinstein I, Suzuki H, and Alkan-Önyüksel H. Development of a novel bioactive formulation of vasoactive intestinal peptide in sterically stabilized liposomes. Pharm Res 14: 362-365, 1997[ISI][Medline].

37.   Séjourné, F, Suzuki H, Alkan-Önyüksel H, Gao XP, Ikezaki H, and Rubinstein I. Mechanisms of vasodilation elicited by VIP in sterically stabilized liposomes in vivo. Am J Physiol Regul Integr Comp Physiol 273: R287-R292, 1997[Abstract/Free Full Text].

38.   Suzuki, H, Gao XP, Olopade CO, and Rubinstein I. Neutral endopeptidase modulates vasoactive intestinal peptide-induced vasodilation in situ. Am J Physiol Regul Integr Comp Physiol 271: R393-R397, 1996[Abstract/Free Full Text].

39.   Suzuki, H, Noda Y, Gao XP, Séjourné F, Alkan-Önyüksel H, Paul S, and Rubinstein I. Encapsulation of vasoactive intestinal peptide into liposomes restores vasorelaxation in hypertension in situ. Am J Physiol Heart Circ Physiol 271: H282-H287, 1996[Abstract/Free Full Text].

40.   Suzuki, H, Noda Y, Paul S, Gao XP, and Rubinstein I. Encapsulation of vasoactive intestinal peptide into liposomes: effects on vasodilation in vivo. Life Sci 57: 1451-1457, 1995[ISI][Medline].

41.   Tornoe, K, Hannibal J, Fahrenkrug J, and Holst JJ. PACAP-(138) as a neurotransmitter in pig pancreas: receptor activation revealed by the antagonist PACAP-(638). Am J Physiol Gastrointest Liver Physiol 273: G436-G446, 1997[Abstract/Free Full Text].

42.   Uddman, R, Luts A, Arimura A, and Sundler F. Pituitary adenylate cyclase activating polupeptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract. Cell Tissue Res 265: 197-201, 1991[ISI][Medline].

43.   Vaudry, D, Gonzalez B, Basille M, Yon L, Flouriner A, and Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to function. Pharmacol Rev 52: 269-324, 2000[Abstract/Free Full Text].

44.   Woodle, MC, Storm G, Newman MS, Jekot JJ, Collins LR, Martin FJ, and Szoka FC, Jr. Prolonged systemic delivery of peptide drugs by long circulating liposomes: illustration with vasopressin in the Brattleboro rat. Pharm Res 9: 260-265, 1992[ISI][Medline].

45.   Xia, WJ, and Önyüksel H. Mechanistic studies on surfactant-induced membrane permeability enhancement. Pharm Res 17: 612-618, 2000[ISI][Medline].

J APPL PHYSIOL 93(4):1377-1383



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI 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 ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsueshita, T.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsueshita, T.
Right arrow Articles by Rubinstein, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online