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Am J Physiol Regul Integr Comp Physiol 276: R1359-R1365, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 5, R1359-R1365, May 1999

Exogenous calmodulin potentiates vasodilation elicited by phospholipid-associated VIP in vivo

Hiroyuki Ikezaki1,2, Manisha Patel3,4, Hayat Önyüksel3,4, Syed R. Akhter1,2, Xiao-Pei Gao1, and Israel Rubinstein1,2

Departments of 1 Medicine, 3 Pharmaceutics and Pharmacodynamics, and 4 Bioengineering, University of Illinois at Chicago, and 2 West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine whether exogenous calmodulin potentiates vasoactive intestinal peptide (VIP)-induced vasodilation in vivo and, if so, whether this response is amplified by association of VIP with sterically stabilized liposomes. Using intravital microscopy, we found that calmodulin suffused together with aqueous and liposomal VIP did not potentiate vasodilation elicited by VIP in the in situ hamster cheek pouch. However, preincubation of calmodulin with liposomal, but not aqueous, VIP for 1 and 2 h and overnight at 4°C before suffusion significantly potentiated vasodilation (P < 0.05). Calmodulin-induced responses were significantly attenuated by calmidazolium, trifluoperazine, and NG-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. The effects of L-NAME were reversed by L- but not D-arginine. Indomethacin had no significant effects on calmodulin-induced responses. Calmodulin had no significant effects on adenosine-, isoproterenol-, acetylcholine-, and calcium ionophore A-23187-induced vasodilation. Collectively, these data indicate that exogenous calmodulin amplifies vasodilation elicited by phospholipid-associated, but not aqueous, VIP in the in situ peripheral microcirculation in a specific, calmodulin active sites-, and nitric oxide-dependent fashion. We suggest that extracellular calmodulin, phospholipids, and VIP form a novel functionally coordinated class of endogenous vasodilators.

microcirculation; vasomotor tone; arteriole; sterically stabilized liposomes; nitric oxide; calmodulin inhibitors; hamster; vasoactive intestinal peptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASOACTIVE INTESTINAL PEPTIDE (VIP) is a 28-amino acid amphipathic peptide localized in perivascular nerves (12, 13, 25, 31, 33). On its release, VIP elicits potent, albeit short-lived, endothelium-dependent and -independent vasodilation in the peripheral microcirculation (4, 10, 15, 16). However, the nature of the local factors that underlie endothelial contribution to VIP vasorelaxation in various microvascular beds are uncertain.

To this end, calmodulin is a ubiquitous extracellular protein expressing hydrophobic binding domains for amphipathic peptides that are exposed on its binding to circulating and plasma membrane phospholipids (5, 6, 11, 21, 26, 31). Extracellular calmodulin has been shown to promote endothelial cell proliferation and elaboration of phlogistic mediators by leukocytes in vitro (7, 14). Stallwood et al. (37) and Andersson et al. (3) showed that calmodulin binds VIP with high affinity in vitro. Importantly, Rubinstein et al. (33) showed recently that calmodulin amplifies the conformational transition of VIP molecule from a predominantly random coil in aqueous solution to an alpha -helix in the presence of phospholipids. The helical conformation is optimal for VIP receptor interaction and protects the peptide from proteolysis (9, 12, 15, 25, 29, 31, 38). On balance, these data suggest that extracellular calmodulin acts as a phospholipid-dependent extracellular signaling molecule that could interact with VIP to modulate vasorelaxation.

The purpose of this study was to begin to address this issue by determining whether exogenous calmodulin potentiates VIP-induced vasodilation in the in situ hamster cheek pouch and, if so, whether this response is amplified by association of VIP with sterically stabilized liposomes (SSL).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of VIP on SSL

We used a method previously described in our laboratory (17, 18, 28, 35, 36, 39, 40). Briefly, egg yolk phosphatidylcholine, egg yolk phosphatidylglycerol, cholesterol, and polyethylene glycol (molecular weight 1,900) linked to distearoyl-phosphatidylethanolamine (molar ratio 5:1:3.5:0.5; phospholipid content 17 mmol) were dissolved and mixed in chloroform. The solvent was evaporated at 45°C in a rotary evaporator under vacuum overnight. The resulting lipid film was rehydrated in 250 µl saline, vortexed, bath sonicated for 5 min, and extruded through stacked polycarbonate filters using the LiposoFast apparatus (pore size 200, 100, and 50 nm; AVESTIN, Ottawa, ON, Canada). Human VIP (0.4 mg) and trehalose (30 mg), a cryoprotectant, were added to the extruded suspension, which was then frozen in acetone-dry ice bath and lyophilized overnight at -46°C under constant pressure (Foreseen 6, Labconco, Kansas City, MO). Thereafter, the lyophilized "cake" was resuspended in 250 µl deionized water. VIP associated with SSL was separated from free VIP by column chromatography (Bio-Gel A-5m, Bio-Rad Laboratories, Richmond, CA) and stored at 4°C until used. Size of SSL was 250 ± 50 nm as determined by quasielastic light scattering (Nicomp model 270 submicron particle sizer, Pacific Scientific, Menlo Park, CA). Phospholipid concentration in SSL was determined by a modified phosphate assay. VIP concentration in SSL was determined by a commercially available ELISA assay kit (Peninsula Laboratories, Belmont, CA) after dissolving SSL with 1% sodium dodecyl sulfate. The recovery was 30% for VIP and 50% for phospholipids, giving a ratio of 0.004 mol VIP/mol of phospholipids.

Preparation of Animals

Adult, male golden Syrian hamsters (n = 66) weighing 128 ± 5 g were anesthetized with pentobarbital sodium (6 mg/100 g body wt ip). A tracheostomy was performed to facilitate spontaneous breathing. A femoral vein was cannulated to inject supplemental anesthesia during the experiment (2-4 mg · 100 g body wt-1 · h-1). A femoral artery was cannulated to record systemic arterial pressure and heart rate. Body temperature was monitored and kept constant (37-38°C) via a feedback controller and heating pad throughout the duration of the experiment.

To visualize the microcirculation of the cheek pouch, we used a method previously described in our laboratory (17, 18, 22, 27, 28, 32-36, 38-40). Briefly, the left cheek pouch was spread over a plastic base plate, and an incision was made in the overlying skin to expose the cheek pouch membrane. The avascular connective tissue layer of the membrane was carefully removed, and an upper plastic chamber was positioned over the base plate. This arrangement forms a triple-layered complex: the base plate, the upper chamber, and the cheek pouch membrane exposed between the two plates. The chamber is connected via a three-way valve to a reservoir that allows continuous suffusion of the cheek pouch chamber with warm (37-38°C) bicarbonated buffer (pH 7.4) bubbled continuously with 95% N2-5% CO2. The chamber is also connected via a three-way valve to an infusion pump (Sage Instruments, Boston, MA) for controlled 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 magnification of ×40. The microscope image was projected through a low-light television camera (Panasonic TR-124 MA, Matsushita Communication Industrial, Yokohama, Japan) on a video screen (Panasonic). The inner diameter of second-order arterioles (42-57 µm), which regulate vascular resistance in the cheek pouch, was determined during the experiment from the video display of the microscope image using a videomicrometer (VIA 100; Boeckler, Tucson, AZ) as previously described in our laboratory (17, 18, 22, 27, 28, 32-36, 38-40). In each animal, the same arteriolar segment was used to measure changes in diameter during the experiment.

Experimental Protocols

Effects of calmodulin on VIP-induced vasodilation. We used two strategies to address this issue. First, we determined the effects of calmodulin suffused together with aqueous VIP and VIP on SSL on arteriolar diameter in the cheek pouch. After suffusing buffer for 45 min (equilibration period), two concentrations of aqueous human VIP (0.5 and 1.0 nmol) were suffused on the cheek pouch for 7 min each in an arbitrary fashion. At least 45 min elapsed between subsequent suffusions of VIP. Once suffusion of VIP was stopped and arteriolar diameter returned to baseline, calmodulin (0.1 nM) was suffused together with VIP (0.5 and 1.0 nmol) for 7 min. In a second group of animals, VIP on SSL (0.01 and 0.1 nmol) was suffused rather than aqueous VIP. These concentrations were previously shown to elicit vasodilation similar in magnitude to that evoked by 0.5 and 1.0 nmol aqueous VIP (17, 18, 35, 36, 39, 40). Arteriolar diameter was determined before and every minute during and after each intervention. In preliminary studies, we found that repeated suffusions of VIP (0.5 and 1.0 nmol) and VIP on SSL (0.01 and 0.1 nmol) in the absence and presence of calmodulin (0.1 nM) for 7 min each were associated with reproducible results. Suffusion of empty SSL for 7 min had no significant effects on arteriolar diameter (1.9 ± 1.1% increase from baseline; n = 4; P > 0.5). Likewise, suffusion of calmodulin (0.1 nM) alone or in the presence of empty SSL for 7 min had no significant effects on arteriolar diameter (1.3 ± 1.1 and 1.4 ± 0.8% increase from baseline, respectively; each group, n = 4; P > 0.5). Suffusion of higher concentrations of calmodulin alone was associated with a significant increase in arteriolar diameter from baseline (data not shown). Suffusion of saline (vehicle) for the entire duration of the experiment was not associated with significant changes in arteriolar diameter.

Second, on the basis of results of the experiments outlined above, we determined whether incubation of calmodulin with aqueous VIP or VIP on SSL before suffusion on the cheek pouch amplifies vasodilation relative to that evoked by aqueous VIP and VIP on SSL alone. Aqueous VIP (0.5 nmol) or VIP on SSL (0.01 nmol) was incubated with calmodulin (0.1 nM) for 1 and 2 h and overnight at 4°C. We previously used this approach to load VIP on SSL (28). After the equilibration period, aqueous VIP (0.5 nmol) was suffused on the cheek pouch using the experimental design outlined above. In preliminary studies, we found that repeated suffusions of aqueous VIP (0.5 nmol) or VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 1 and 2 h and overnight at 4°C for 7 min each were associated with reproducible results. Suffusion of empty SSL incubated with calmodulin (0.1 nM) for 1 and 2 h and overnight at 4°C for 7 min had no significant effects on arteriolar diameter (1.3 ± 0.6, 1.4 ± 0.6, and 2.5 ± 1.6% increase from baseline, respectively; each group, n = 4; P > 0.5). Suffusion of aqueous VIP (0.5 nmol) incubated alone for 1 and 2 h and overnight at 4°C for 7 min elicited vasodilation similar to that evoked by freshly prepared aqueous VIP (0.5 nmol). The concentrations of aqueous VIP, VIP on SSL, and calmodulin used in these experiments were based on previous studies in our laboratory and reports in the literature (1, 17-20, 24, 35, 36, 39, 40).

Mechanisms of Calmodulin-Induced Responses

Effects on other vasodilators. The purpose of this study was to determine whether calmodulin potentiation of VIP on SSL-induced vasodilation is specific. To this end, we determined the effects of calmodulin on vasodilation elicited by adenosine (0.1 µM), isoproterenol (0.01 µM), acetylcholine (1.0 µM), and calcium ionophore A-23187 (1.0 µM), compounds that elicit vasodilation in the cheek pouch through distinct pathways (17, 18, 32, 36, 39), using the experimental design outlined above, except that each agonist was now incubated with calmodulin (0.1 nM) for 2 h at 4°C before suffusion. We chose this incubation period on the basis of results of studies using calmodulin incubated with VIP on SSL outlined above. In preliminary studies, we found that the concentrations of adenosine, isoproterenol, acetylcholine, and calcium ionophore A-23187 used in these experiments elicited vasodilation of a magnitude similar to that evoked by VIP on SSL (0.1 nmol) in a reproducible fashion.

Effects of calmodulin inhibitors. The purpose of this study was to determine whether calmodulin potentiation of VIP on SSL-induced vasodilation is active sites dependent. We determined the effects of calmidazolium and trifluoperazine, two structurally distinct calmodulin inhibitors (1, 19, 24), on calmodulin-induced responses. VIP (1.0 nmol), VIP on SSL (0.01 and 0.1 nmol), and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C were suffused before and 30 min after suffusion of calmidazolium or trifluoperazine (each 10 µM). In preliminary studies, we found that suffusion of calmidazolium and trifluoperazine (each 10 µM) alone for 37 min was associated with no significant change in arteriolar diameter (1.3 ± 0.4 and 0.8 ± 0.8% increase from baseline, respectively; each group, n = 4; P > 0.5). Suffusion of higher concentrations of both inhibitors was associated with vasoconstriction (data not shown). Suffusion of DMSO (0.05%), the vehicle of calmidazolium, alone was associated with no significant change in arteriolar diameter (1.4 ± 0.8% increase from baseline; n = 4; P > 0.5). In addition, suffusion of DMSO (0.05%) for 30 min before and during suffusion of VIP (1.0 nmol), VIP on SSL (0.01 and 0.1 nmol), and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C for 7 min had no significant effects on agonist-induced vasodilation relative to that evoked by each agonist alone. The concentrations of calmidazolium and trifluoperazine used in these experiments were based on preliminary studies and reports in the literature (1, 19, 24).

Effects of nitric oxide synthase inhibition. The purpose of this study was to determine whether calmodulin potentiation of VIP on SSL-induced vasodilation is mediated, in part, by the L-arginine-nitric oxide (NO) biosynthetic pathway. VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C was suffused before and 30 min after suffusion of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, or NG-nitro-D-arginine methyl ester (D-NAME; each 10 µM) as outlined above. In another group of animals, L-arginine, the substrate for NO synthase, or D-arginine (each 100 µM) was suffused together with L-NAME 30 min before and during suffusion of VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C. In preliminary studies, we found that suffusion of L-NAME (10 µM) and L-arginine (100 µM) alone or together had no significant effects on arteriolar diameter and systemic arterial pressure. The concentrations and method of administration of L-NAME, D-NAME, L-arginine, and D-arginine used in these experiments were based on previous studies in our laboratory (2, 32, 34, 36, 39).

Effects of indomethacin. The purpose of this study was to determine whether calmodulin potentiation of VIP on SSL-induced vasodilation is mediated, in part, by cyclooxygenase products of arachidonic acid metabolism. Aqueous VIP (1.0 nmol), VIP on SSL (0.01 and 0.1 nmol), and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C were suffused before and 30 min after intravenous infusion of indomethacin (10 mg/kg) using an infusion pump (Sage Instruments; final volume 1 ml) as outlined above. In preliminary studies, we found that indomethacin (10 mg/kg iv) had no significant effects on arteriolar diameter and systemic arterial pressure. The concentration and mode of administration of indomethacin used in these experiments were based on previous studies in our laboratory and a report in the literature (17, 30, 34, 36).

Chemicals and drugs. Egg yolk phosphatidylcholine, egg yolk phosphatidylglycerol, cholesterol, trehalose, adenosine, isoproterenol, acetylcholine, calcium ionophore A-23187 hemimagnesium, trifluoperazine, DMSO, L-NAME, D-NAME, L-arginine, and D-arginine were obtained from Sigma (St. Louis, MO). Human VIP was obtained from American Peptide Company (Sunnyvale, CA). Calmodulin (bovine brain) and calmidazolium were obtained from Calbiochem (La Jolla, CA). Calmidazolium was dissolved and diluted in DMSO. Indomethacin was dissolved in sodium bicarbonate. All drugs, except calmidazolium, were diluted in saline to the desired concentrations on the day of the experiment.

Data and statistical analyses. When a compound was suffused on the cheek pouch, we determined the maximal change in arteriolar diameter and used it as the response to that compound in each animal. Arteriolar diameter was expressed as the ratio of experimental to control diameter, with control diameter normalized to 100%, to account for intra- and interanimal variability. Data are expressed as means ± SE, except for the size of VIP on SSL and body weight, which were expressed as means ± SD because these data are not used for comparison between experimental groups. Statistical analysis was performed using repeated-measures analysis of variance with Newman-Keuls multiple-range post hoc test to detect values that were different from control values. A P value <0.05 was considered statistically significant; n is given as the number of experiments, with each experiment representing a separate animal.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mean arterial pressure was 102 ± 2 mmHg at the start and 98 ± 1 mmHg at the conclusion of the experiments (n = 66; P > 0.5).

Effects of Calmodulin on VIP-Induced Vasodilation

Suffusion of aqueous VIP elicited significant, concentration-dependent increase in arteriolar diameter (Fig. 1; each group, n = 4; P < 0.05). Suffusion of calmodulin (0.1 nM) together with VIP had no significant effects on VIP-induced vasodilation (Fig. 1; each group, n = 4; P > 0.5). Suffusion of VIP on SSL also elicited significant, concentration-dependent increases in arteriolar diameter (Fig. 1; each group, n = 4; P < 0.05). Suffusion of calmodulin (0.1 nM) together with VIP on SSL had no significant effects on VIP on SSL-induced responses (Fig. 1; each group, n = 4; P > 0.5).


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  		Fig. 1.   Effects of suffusion of aqueous vasoactive intestinal peptide (VIP) and VIP on sterically stabilized liposomes (SSL) on arteriolar diameter in in situ hamster cheek pouch in absence and presence of calmodulin (CaM). Values are means ± SE; each group, n = 4. * P < 0.05 compared with baseline.

By contrast, incubation of VIP on SSL (0.01 nmol) with calmodulin (0.1 nM) for 1 and 2 h and overnight at 4°C significantly potentiated VIP-induced vasodilation (Fig. 2; each group, n = 4; P < 0.05). Incubation of aqueous VIP (0.5 nmol) with calmodulin (0.1 nmol) for 1 and 2 h and overnight at 4°C had no significant effects on VIP-induced responses (Fig. 2; each group, n = 4; P > 0.5).


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  		Fig. 2.   Effects of suffusion of aqueous VIP and VIP on SSL on arteriolar diameter in in situ hamster cheek pouch alone and after incubation with CaM for 1 and 2 h and overnight at 4°C. Values are means ± SE; each group, n = 4. * P < 0.05 compared with baseline; dagger  P < 0.05 compared with VIP on SSL alone.

Mechanisms of Calmodulin-Induced Responses

Effects on other vasodilators. Calmodulin (0.1 nM) suffused together with adenosine (0.1 µM), isoproterenol (0.01 µM), acetylcholine (1.0 µM), and calcium ionophore A-23187 (1.0 µM) or incubated together with each agonist for 2 h at 4°C before suffusion had no significant effects on agonist-induced vasodilation (Table 1; each group, n = 4; P > 0.5).

                              
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  		Table 1.   Effects of calmodulin on agonist-induced vasodilation in the in situ hamster cheek pouch

Effects of calmodulin inhibitors. Calmidazolium (10 µM) significantly attenuated vasodilation elicited by aqueous VIP (1.0 nmol), VIP on SSL (0.01 and 0.1 nmol), and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C (Fig. 3A; P < 0.05). Trifluoperazine (10 µM) also significantly attenuated vasodilation elicited by VIP on SSL (0.01 and 0.1 nmol) and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C (Fig. 3B; P < 0.05). However, it had no significant effects on aqueous VIP (1.0 nmol)-induced responses (Fig. 3B; each group, n = 4; P > 0.5).


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  		Fig. 3.   Effects of calmidazolium (A; CMZ) and trifluoperazine (B; TFP) on arteriolar diameter in in situ hamster cheek pouch elicited by aqueous VIP, VIP on SSL, and VIP on SSL incubated with CaM (0.1 nM) for 2 h at 4°C. Values are means ± SE; each group, n = 4. * P < 0.05 compared with baseline; dagger P < 0.05 in absence of CMZ and TFP, respectively.

Effects of NO synthase inhibition. L-NAME (10 µM) significantly attenuated vasodilation elicited by VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C (Fig. 4; each group, n = 4; P < 0.05). D-NAME (10 µM) had no significant effects on vasodilation elicited by VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C (Fig. 4; each group, n = 4; P > 0.5). L-Arginine, but not D-arginine (each 100 µM), abrogated L-NAME (10 µM)-induced responses (0.1 nM) for 2 h at 4°C (Fig. 4; each group, n = 4).


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  		Fig. 4.   Effects of NG-nitro-L-arginine methyl ester (L-NAME), D-NAME, and L-NAME together with L-arginine or D-arginine on arteriolar diameter in in situ hamster cheek pouch elicited by VIP on SSL incubated with CaM (0.1 nM) for 2 h at 4°C. Values are means ± SE; each group, n = 4. * P < 0.05 compared with baseline; dagger P < 0.05 in comparison to VIP on SSL incubated with CaM (0.1 nM) for 2 h at 4°C alone.

Effects of indomethacin. Indomethacin has no significant effects on vasodilation elicited by aqueous VIP (1.0 nmol), VIP on SSL (0.01 and 0.1 nmol), and VIP on SSL (0.01 nmol) incubated with calmodulin (0.1 nM) for 2 h at 4°C (Table 2; each group, n = 4; P > 0.5).

                              
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  		Table 2.   Effects of indomethacin on VIP-induced vasodilation in the in situ hamster cheek pouch


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are several new findings of this study. We found that calmodulin, a ubiquitous extracellular regulatory protein that interacts with phospholipids and amphipathic peptides (5-7, 11, 14, 21, 26) at picomolar concentrations detected in biological fluids (20), amplified vasodilation elicited by phospholipid (liposome)-associated, but not aqueous, VIP in the in situ hamster cheek pouch. This response was observed only when calmodulin was preincubated with liposomal VIP before suffusion and was calmodulin active sites-dependent because calmidazolium and trifluoperazine, two structurally distinct calmodulin inhibitors (1, 19, 24), significantly attenuated calmodulin-induced responses. Suffusion of calmodulin alone or calmodulin preincubated with aqueous VIP or empty SSL, at concentrations used in this study, had no significant effects on arteriolar diameter.

Calmodulin potentiation of liposomal VIP-induced responses was specific because arteriolar diameter returned to baseline once suffusion of calmodulin preincubated with liposomal VIP was stopped and because calmodulin preincubated with adenosine, isoproterenol, acetylcholine, and calcium ionophore A-23187, compounds that elicit vasodilation in the cheek pouch by distinct mechanisms and intracellular signal transduction pathways (17, 18, 32, 36, 39), had no significant effects on agonist-induced responses. Importantly, the potentiating effects of calmodulin on liposomal VIP-induced vasodilation were not related to local elaboration of vasodilator prostaglandins because indomethacin, at a concentration that inhibits cyclooxygenase in the cheek pouch (30), had no significant effects on calmodulin-induced responses.

Taken together, these data indicate that VIP, an amphipathic peptide released from nerve endings in the peripheral microcirculation (13, 15, 16), interacts with extracellular calmodulin in the presence of phospholipids to amplify vasodilation in vivo. This novel extracellular regulatory pathway is time dependent and requires the presence of all three components, calmodulin, phospholipids, and VIP, in the extracellular milieu to be functionally expressed.

Previous work from our laboratory showed that, unlike aqueous VIP, phospholipid-associated VIP (liposomal and micellar) elicits potent and prolonged vasodilation in the cheek pouch that is transduced by the L-arginine-NO biosynthetic pathway (17, 18, 27, 28, 35, 36, 38-40). The results of this study support and extend these observations by showing that calmodulin potentiation of liposomal VIP-induced vasodilation in the cheek pouch is transduced by the L-arginine-NO biosynthetic pathway provided sufficient time is allowed for calmodulin to interact with liposomal VIP (8, 23).

The mechanisms underlying the molecular interactions between extracellular calmodulin, phospholipids, and VIP that amplify vasodilation were not elucidated in this study. Conceivably, circulating and plasma membrane phospholipids could evoke conformational transition of the VIP molecule from a predominantly random coil to an alpha -helix and expose hydrophobic domains in the calmodulin molecule (3, 5, 6, 11, 20, 26, 37). These domains recognize and bind to the alpha -helix component of the VIP molecule with high affinity and could activate the L-arginine-NO biosynthetic pathway in resistance arterioles (2, 8, 11, 12, 15, 19, 21-23, 25, 31, 32, 36). The results of this study support, in part, this hypothesis because exogenous calmodulin had no significant effects on vasodilation elicited by aqueous (random coil) VIP in the cheek pouch. Clearly additional studies using molecular, biochemical, and cell biology techniques are warranted to support or refute this hypothesis.

Perspectives

This study unravels a novel extracellular mechanism that regulates vasorelaxation in the peripheral microcirculation, consisting of hydrophobic interactions between extracellular calmodulin, phospholipids, and VIP that promote potent NO-dependent vasodilation. We postulate that this control system could be impaired in certain diseases characterized by NO-dependent vasomotor dysfunction, such as hypertension, heart failure, and impotence (23).

In summary, we found that exogenous calmodulin amplifies vasodilation elicited by phospholipid-associated, but not aqueous, VIP in the in situ peripheral microcirculation in a specific, calmodulin active sites- and NO-dependent fashion. We suggest that extracellular calmodulin, phospholipids, and VIP form a novel, functionally coordinated class of endogenous vasodilators.


    ACKNOWLEDGEMENTS

This study was supported, in part, by grants from the National Institutes of Health (DE-10347) and American Heart Association of Metropolitan Chicago. Dr. Rubinstein is a recipient of a Research Career Development Award from the National Institutes of Health (DE-00386) and a University of Illinois Scholar Award.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. Rubinstein, Dept. of Medicine (M/C 787), Univ. of Illinois at Chicago, 840 South Wood St., Chicago, IL 60612-7323 (E-mail: IRubinst{at}uic.edu).

Received 29 June 1998; accepted in final form 29 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahlijanian, M. K., and D. M. F. Cooper. Antagonism of calmodulin-stimulated adenyl cyclase by trifluoperazine, calmidazolium and W-7 in rat cerebellar membranes. J. Pharmacol. Exp. Ther. 241: 407-414, 1987[Abstract/Free Full Text].

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

3.   Andersson, M., M. Carlquist, M. Maletti, and J.-C. Marie. Simultaneous solubilization of high-affinity receptors for VIP and glucagon and of a low-affinity protein for VIP, shown to be identical to calmodulin. FEBS Lett. 318: 35-40, 1993[Medline].

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Am J Physiol Regul Integr Compar Physiol 276(5):R1359-R1365
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