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Am J Physiol Regul Integr Comp Physiol 280: R713-R720, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 3, R713-R720, March 2001

Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin

Cho Y. Pang1,2,3, Huai Xu1, Ning Huang1, Christopher R. Forrest1,2, Thérèse M. Perréault4, and Peter C. Neligan1,2

1 Research Institute, The Hospital for Sick Children, and Departments of 2 Surgery and 3 Physiology, University of Toronto, Toronto, Ontario M5G 1X8, and 4 Division of Newborn Medicine, The Montreal Children's Hospital, Montreal, Quebec H3H 1P3, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 × 10-10 M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 × 16 cm) were perfused with Krebs buffer equilibrated with 95% O2 and 5% CO2 at 37°C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 × 10-10 M) significantly amplified the concentration-dependent (10-7-10-5 M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10-8-10-6 M) skin vasoconstrictor effect of the thromboxane A2 mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 × 10-6 M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10-7 M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca2+ in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 × 10-10 M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 × 10-6 M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca2+ in smooth muscle cells.

skin vasospasm; endothelin-1; protein kinase C; Ca2+ sensitivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN (ET)-1-(1-21) is a 21-amino acid peptide that is synthesized and released by endothelial cells. ET-1 is the most potent vasoconstrictor and the predominant isoform expressed in vasculature, and it also has a long-lasting pressor effect (16, 17, 41). ET-1 has been implicated in cardiovascular disease, such as myocardial infarction, cardiac failure, essential and pulmonary hypertension, Buerger's disease, and Raynaud's phenomenon (8, 12, 21, 24), partly because the circulating levels of ET-1 in these disease states have been observed to increase by 2- to 10-fold (4, 8, 24). It remains to be established whether ET-1 is primarily involved in the development of these cardiovascular diseases, because the elevated levels of ET-1 in these disease states are much lower than the levels of ET-1 required to induce significant vasoconstrictor effects in isolated blood vessels. However, there is in vitro evidence to indicate that ~80% of the total amount of ET-1 synthesized by endothelial cells was found on the abluminal side, thus indicating that high local levels of ET-1 may be present at the vascular smooth muscle in pathologic states (39).

On the other hand, threshold or low levels of ET-1 may play an important role in the pathogenesis of cardiovascular diseases if ET-1 can amplify the effects of other vasoconstrictor substances that are found at much higher elevated circulating levels than ET-1 or liberated locally from sympathetic nerve endings, such as norepinephrine (NE), or released from activated platelets, such as thromboxane A2 (TxA2) or serotonin (5-HT). Indeed, threshold or low levels of ET-1 have been observed to amplify the in vitro vasoconstrictor effect of 5-HT in human internal mammary artery, coronary artery, umbilical artery, and placenta and in rabbit and pig aorta and rat mesenteric artery (5, 11, 14, 27, 28, 42). Furthermore, subthreshold or threshold levels of ET-1 have also been observed to amplify the in vitro vasoconstrictor effect of NE in human mammary artery and rabbit aorta (15, 42). The amplification effect of ET-1 on TxA2-induced vasoconstriction has been discussed (14), but there are no published data available thus far on the effect and mechanism of action of ET-1 in augmentation of TxA2-induced vasoconstriction.

Of particular interest to us is the potential interaction of ET-1 and TxA2 in the pathogenesis of skin vasospasm. Specifically, autogenous skin or skin and muscle transplantation (i.e., cutaneous or musculocutaneous free flap surgery) is routinely used for coverage of large deep wounds. A cutaneous or musculocutaneous flap is harvested from a distant donor site of the body and is transferred to cover the wound, with vascular anastomosis performed at the recipient site to reestablish blood supply (7). Despite advances in microsurgical technique and judicious donor site selection and recipient site preparation, flap failure associated with vasospasm and thrombosis still occur at a rate of 5-10% even in large medical centers (1). Vasospasm can occur intraoperatively, shortly after anastomosis, or within 72 h postoperatively. Vasospasm plays an important role in the pathogenesis of thrombosis, causing partial or total flap ischemic necrosis (23, 40). The mechanism of skin vasospasm in free flap surgery is unclear. It seems that mechanical stretch or trauma may induce a myogenic response, causing vasospasm. Surgical trauma may induce local vasospasm by stimulating the sympathetic nerve ending to release NE (30). The synthesis and release of endothelium-derived relaxing factors, such as prostacyclin and nitric oxide, may be reduced or the synthesis and release of endothelium-derived contracting factors, such as TxA2 and ET-1, may be increased (2, 3, 7, 9, 13, 18, 22). Last but not least, reduced local blood flow due to vasospasm may also increase the thrombogenic nature of the suture lines of the vascular anastomosis. This may in turn promote platelet release of vasoconstrictive and prothrombotic substances such as NE, TxA2, and 5-HT (19). We previously demonstrated that ET-1 is a potent and long-acting vasoconstrictor in pig and human skin (20, 31, 32). Here, we planned to demonstrate that low concentrations of ET-1 may also contribute to the pathogenesis of skin vasospasm by amplifying the vasoconstrictor effect of other vasoactive substances. To this end, we studied the amplification effect and mechanism of action of ET-1 in NE- and U-46619-induced vasoconstriction in pig skin. U-46619 was chosen because it is a long-acting TxA2 mimetic and there are no published data available thus far to demonstrate the potentiation effect and mechanism of action of ET-1 on the vasoconstrictor effect of TxA2, especially in the skin. The isolated perfused pig buttock skin flap model was chosen for this project because the skin vasculature of this skin flap model closely resembles that of the human skin free flap (6) and we previously used this isolated perfused pig buttock skin flap model to study skin vascular reactivities and mechanism of action in response to intra-arterial drug infusion (10, 29, 31, 32). An isolated perfused pig skin flap model has been described and validated by other investigators (26, 34-36). Skin tissue viability was assessed by monitoring perfusate flow rate, perfusion pressure, pH, glucose use, lactate production, and enzyme leakage (26, 36). Skin viability up to 24 h was confirmed through biochemical studies and extensive light and electron microscopic histological studies (34). In addition, the vascular responses of this skin flap model to classical pharmacologic agents have been assessed and were found to be similar to classic in vitro and in vivo models (35).

Using an isolated perfused pig skin flap model in the present study, we demonstrated that a low concentration of ET-1 (8 × 10-10 M) can amplify the vasoconstriction effects of NE and U-46619 in the skin vasculature. In addition, we have demonstrated for the first time that the mechanism of ET-1 in amplification of U-46619-induced vasoconstriction in the skin may involve activation of intracellular protein kinase C (PKC) and increase in sensitivity of the contractile apparatus to extracellular Ca2+. Last but not least, the amplification effect of ET-1 on U-46619-induced skin vasoconstriction is independent of endogenous cyclooxygenase products.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Procedures

Castrated pigs (20 ± 2 kg; mean ± SD) were used. Skin flaps were harvested under general anesthesia induced by intramuscular ketamine (25 mg/kg) and intravenous pentobarbital sodium (20-25 mg/kg). General anesthesia was maintained by intravenous infusion of isotonic saline (2 ml/min) containing pentobarbital sodium (0.5 mg/kg). A 6 × 16-cm skin flap based on the deep circumflex iliac neurovascular bundle was outlined on both sides of the buttock. These marked skin flaps were incised and completely undermined with all musculocutaneous blood vessels (perforators) tied and/or cauterized carefully. The circumflex iliac neurovascular bundle was dissected and formed the vascular pedicle (4-5 cm) of the island buttock skin flap. All side branches of blood vessels of the pedicle were ligated with 3-0 silk sutures and cauterized. Finally, the proximal end of the neurovascular pedicle of the flap was tied with a 2-0 silk suture and then transected. The arterialized island buttock skin flap was freed and used for in vitro perfusion. The pig was killed with an overdose of intravenous pentobarbital sodium (100 mg/kg). This animal protocol was approved by The Hospital for Sick Children Animal Care Committee.

Skin Flap Preparation for in Vitro Perfusion

The skin flap was wrapped around a plastic tube (22 cm in length and 1.2 cm in diameter), and the longitudinal edges of the flap were sewn together with 3-0 silk sutures to form a tubed flap. From our previous experience, inclusion of this tube in the tubed skin flap significantly reduced edema formation during 4-5 h of in vitro perfusion, and water retention was reduced to <10% (10, 29, 31, 32). The circumflex iliac artery and one of its veins were cannulated with a 20- and 18-gauge angiocatheter, respectively, for skin flap perfusion. The warm ischemic time required for construction of a tubed skin flap and cannulation was 20-30 min. We previously demonstrated that this pig skin flap model could tolerate 2 h of primary and 10 h of secondary ischemia without skin necrosis (13). Clinically, the warm ischemic time for free flap surgery is 40-45 min. Therefore, it is unlikely that skin ischemia-reperfusion injury could have taken place in our isolated perfused skin flap model.

Skin Flap Perfusion Technique

Modified Krebs-Henseleit buffer of the following composition (mM) was used for perfusate: 100 NaCl, 4.60 KCl, 1.10 NaH2PO4, 1.20 MgSO4, 2.25 CaCl2, 30 NaHCO3, 11 glucose, and 2 D-mannitol. Bovine serum albumin (Cohn fraction V) was added to the buffer (65 g/l), which was stirred and filtered (Whatman 44) before use. Ca2+-free buffer contained 2 mM EGTA. Similar perfusate was used by other investigators in the isolated perfused pig skin flap model (26, 34-36). In the present projects, Krebs buffer instead of whole blood was chosen as perfusate to avoid confounding factors in studying the mechanism of action of ET-1 in amplification of U-46619-induced vasoconstriction in skin vasculature.

The commercially available Two-Ten Perfuser (MX International, Aurora, CO) equipped with two reservoirs and a pump with adjustable rates (model 7014, Cole-Palmer Instrument, Vernon Hills, IL) was used as a perfusion apparatus. The perfusate was equilibrated in the reservoirs with 95% O2 and 5% CO2 at 38°C and pH 7.35-7.40. The perfusate was kept at 37°C. A three-way connector that linked the tubing from the peristaltic pump to the arterial angiocatheter of the flap permitted a parallel tubing to be connected to a pressure transducer (AB High Performance Pressure Transducer, Data Instrument, Lexington, KY). The transducer output was displayed continuously on a digital monitor (Trendicator II 621A, Doric Scientific, San Diego, CA) and a chart recorder (Lineacorder WR3101, Graphtec). The pump was adjusted to produce a basal perfusion pressure of 36-40 mmHg. Drugs to be tested were infused into the perfusate through a side arm shortly before the perfusate entering the arterial angiocatheter of the skin flap. A thermistor probe (YSI Series 400, Yellow Springs Instrument, Yellow Springs, OH) connected to a microcomputer thermometer (series 084 202, Cole Parmer Instrument) was positioned on the surface of the longitudinal midpoint of the skin flap for continuous monitoring of surface skin temperature, which was kept at ~34°C.

A baseline perfusion pressure of 36-40 mmHg was selected, because our past experiments revealed that the pig skin flap was well perfused and oxygenated (10, 29, 31, 32). The basal perfusion pressure used by other investigators for perfusion of rabbit ears was 35.8 ± 3.5 mmHg (33). From our past experience, we also noticed that the weights of the 6 × 16-cm buttock skin flaps in pigs weighing 17-22 kg were quite uniform (51 ± 3 g). A pump rate of ~2.0 ml/min would produce a baseline perfusion pressure of 36-40 mmHg. In all the studies reported here, a 45-min stabilization period was allowed to establish a steady baseline perfusion pressure at a constant flow rate. Unless otherwise stated, ET-1 and other drugs used as inhibitors or antagonists were infused continuously, starting 45 min before infusion of an agonist. Cumulative concentration-dependent curves were constructed by stepwise addition of the constrictor substances to the perfusate buffer. Each increment was made only after the response for the preceding concentration of drug had stabilized, ~15 min for NE and U-46619.

Surface Dermofluorometry Technique for Assessment of Skin Perfusion

The dermofluorometry technique for indirect assessment of in vivo dermal perfusing has been validated against the radioactive microsphere technique in the pig skin (38). Dermofluorometry has also been applied to an isolated perfused human skin flap model in vitro (20). Specifically, confluent circles of 1-cm diameter were marked along the longitudinal midline of the skin flap surface. After the 45-min stabilization period, the background fluorescence in each circular skin area was measured (fluorescence units) using a dermofluorometer (Fluorescan unit; Santa Barbara Technology, Santa Barbara, CA). Fluorescence dye (Diofluor, fluorescein sodium, 100 mg/ml, Dioptic Laboratories, Markham, ON, Canada) with a final concentration of 3 × 10-5 M was then infused into the skin flap for 5 min, and fluorescence in each circle was measured again. A washout period of 15 min was allowed, and the background fluorescence was taken again. After the perfusion pressure had stabilized subsequent to drug infusion for the study of skin vascular reactivity, fluorescence dye infusion was repeated, and skin fluorescence was measured again. The difference in fluorescence units for each circular skin area between the background and post-fluorescence dye infusion was defined as the net fluorescence unit for that area. The total dye fluorescence is the sum of all net fluorescence units measured from all circular skin areas along the midline of the skin flap (20, 38).

Biochemicals

All reagents and drugs were purchased from Sigma Chemical (Oakville, ON, Canada), except the following: ET-1-(1-21) from Peptides International (Louisville, KY); fluorescence dye (Diofluor, fluorescein sodium 100 mg/ml) from Dioptic Laboratories; NE from SABEX Pharmaceutical (Boucherville, Quebec, Canada); phorbol 12,13-dibutyrate (PDBu) from Biomol Research Laboratories (Plymouth Meeting, PA); and U-46619 from Cayman (Ann Arbor, MI).

Purified water (Milli-Q Water System, Bedford, MA) was used for making solutions and perfusion buffer. ET-1 stock solution (10-4 M) was made with 0.1% acetic acid and stored at -80°C until use. PDBu and U-46619 were each dissolved in 200 µl of DMSO and indomethacin in 200 µl of ethanol before adding to the buffer perfusion. This amount of DMSO and ethanol did not affect the baseline perfusion pressure of the isolated perfused skin flaps.

Experimental Protocols

The following studies were undertaken to investigate whether a low concentration of ET-1 (8 × 10-10 M) could potentiate the vasoconstrictor effect of NE and U-46619 in pig skin and, if so, what possible mechanism may be involved. ET-1 concentration of 8 × 10-10 M was chosen because our previous data indicated that the mean threshold concentration of ET-1 in this isolated perfused pig skin flap model was 5 × 10-10 M (31, 32). In our preliminary study, it was observed that 8 × 10-10 M of ET-1 increased the baseline perfusion pressure by 6-8 mmHg, and this was tested over 120 min. This low concentration of ET-1 was infused into the pig skin flap starting at 45 min before NE or U-46619 infusion, and the new baseline with ET-1 was used for calculation of increase in perfusion pressure. A low concentration of 10-9 M of ET-1 was used by other investigators to study the amplification effect of ET-1 on NE-induced contraction in human mammary rings (42).

Protocol 1: to investigate the amplification effect of a low concentration of ET-1 (8 × 10-10 M) on NE and U-46619-induced skin vasoconstriction. Cumulative concentration-dependent (10-7-10-5 M) vasoconstrictor effects of NE and U-46619 on perfusion pressure were studied in the absence and presence of 8 × 10-10 M of ET-1 in pig skin flaps perfused with normal Krebs buffer.

In a separate study, the dermofluorometry technique was used to corroborate the observation of skin vascular reactivities assessed by measurement of perfusion pressure observed in the above study. Specifically, the vasoconstrictor effect of U-46619 on skin perfusion in the absence and presence of 8 × 10-10 M of ET-1 in pig skin flaps perfused with normal Krebs buffer was assessed using the dermofluorometry technique. Total dye fluorescence was assessed at the end of stabilization period (baseline), 15-min infusion of vehicle, and 15-min infusion of U-46619 in the absence and presence of 8 × 10-10 M of ET-1.

Protocol 2: to investigate the role of PKC in the amplification effect of a low concentration of ET-1 on U-46619-induced skin vasoconstriction. The cumulative concentration-dependent effect of U-46619 (10-8-10-6 M) on perfusion pressure in pig skin flaps perfused with Krebs buffer was studied in the absence and presence of 8 × 10-10 M ET-1 and in the presence of both 8 × 10-10 M of ET-1 and the PKC inhibitor chelerythrine (5 × 10-6 M).

In a separate study, the concentration-dependent vasoconstriction effect of U-46619 (10-8-10-6M) on perfusion pressure in pig skin flaps was investigated 1) alone, 2) in the presence of 8 × 10-10 M of ET-1, and 3) in the presence of a low concentration of the PKC activator PDBu (10-7 M). The threshold concentration of PDBu in past experiments with this skin flap model was 5 × 10-8 M (32).

Protocol 3: to investigate the role of Ca2+ in the amplification effect of a low concentration of ET-1 on U-46619-induced skin vasoconstriction. The Krebs buffer used in this study contained various concentrations of Ca2+ (10-5-10-2 M). The vasoconstrictor effect of U-46619 (5 × 10-7 M) on perfusion pressure in pig skin flaps was studied in the absence and presence of 8 × 10-10 M of ET-1 at various concentrations of Ca2+ in the perfusion buffer.

Protocol 4: to investigate the role of endogenous cyclooxygenase products (e.g., PGH2/TxA2) in the amplification effect of a low concentration of ET-1 on U-46619-induced skin vasoconstriction. In this study, the normal Krebs buffer used for skin flap perfusion contained the cyclooxygenase inhibitor indomethacin (5 × 10-6 M) and the nitric oxide synthase inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME, 5 × 10-6 M). The cumulative concentration-dependent effect of U-46619 (10-8-10-6 M) on perfusion pressure in pig skin flaps was studied in the absence and presence of 8 × 10-10 M of ET-1. L-NAME was added because inhibition of cyclooxygenase may increase nitric oxide synthase activity.

Statistics

All values are expressed as means ± SE. The number of observations and the specific statistical tests used in each study are indicated in the legend of each figure. Statistical significance was set at P <=  0.05 for all tests.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Amplification Effect of a Low Concentration of ET-1 on the Vasoconstrictor Effect of NE and U-46619 in Pig Skin

ET-1 (8 ×10-10 M) increased the baseline perfusion pressure by <6 mmHg. This new baseline was used for calculation of increase in perfusion pressure induced by NE and U-46619. Both NE and U-46619 elicited an increase in perfusion pressure in pig skin flaps in a concentration-dependent manner (Figs. 1 and 2). This increase in skin perfusion pressure induced by NE and U-46619 was significantly higher (P < 0.05) in the presence of a low concentration (8 × 10-10 M) of ET-1.


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  		Fig. 1.   Cumulative concentration-dependent skin vasoconstrictor effect of norepinephrine (NE) in the absence and presence of 8 × 10-10 M of endothelin-1 (ET-1). The increase in perfusion pressure elicited by NE was significantly higher (P < 0.05; n = 5) in the presence of a low concentration of ET-1 compared with NE; ANOVA with repeated measures. ET-1 alone increased perfusion pressure by <6 mmHg, and the new baseline was used for calculation of increase in perfusion pressure.



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  		Fig. 2.   Cumulative concentration-dependent skin vasoconstrictor effect of U-46619 in the absence and presence of 8 × 10-10 M of ET-1. The increase in perfusion pressure elicited by U-46619 was significantly higher (P < 0.05; n = 6) in the presence of a low concentration of ET-1 compared with U-46619 alone; ANOVA with repeated measures. ET-1 alone increased perfusion pressure by <6 mmHg, and the new baseline was used for calculation of increase in perfusion pressure.

The effect of U-46619 on skin perfusion was further assessed using the dermofluorometry technique. The total dye fluorescence in the vehicle-treated skin flaps (control) was similar (103 ± 6%) to that of the baseline (Fig. 3). Infusion of 5 × 10-7 M U-46619 significantly reduced (P < 0.05) the total dye fluorescence to 57 ± 8% of the baseline. In the presence of a low concentration of ET-1 (8 × 10-10 M), this same concentration of U-46619 further reduced the total dye fluorescence to 32 ± 4% (P < 0.05) of the baseline (Fig. 3).


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  		Fig. 3.   Amplification effect of a low concentration of ET-1 (8 × 10-10 M) in U-46619-induced (5 × 10-7 M) decrease in skin perfusion assessed by the dermofluorometry technique. One-way ANOVA followed by Duncan's multiple-range test was used for multiple comparisons of means (P < 0.05; n = 4). Means without a common letter are significantly different: a > b > c.

Role of PKC in the Amplification Effect of a Low Concentration of ET-1 on U-46619-Induced Skin Vasoconstriction

Again, the concentration-dependent increase in perfusion pressure elicited by U-46619 was significantly higher (P < 0.05) in the presence of a low concentration of ET-1 (8 × 10-10 M) compared with U-46619 alone (Fig. 4). Pretreatment with the PKC inhibitor chelerythrine (5 × 10-6 M) completely blocked the amplification effect of ET-1 on U-46619-induced increase in perfusion pressure (Fig. 4). Infusion of PKC alone for 45 min did not affect the baseline perfusion pressure.


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  		Fig. 4.   Effect of the protein kinase C (PKC) inhibitor chelerythrine on the amplification effect of a low concentration of ET-1 in U-46619-induced skin vasoconstriction. The concentration-dependent increase in perfusion pressure elicited by U-46619 was significantly higher (P < 0.05) in the presence of a low concentration of ET-1 (8 × 10-10 M) compared with U-46619 alone. This amplification effect of ET-1 on U-46619-induced increase in perfusion pressure was not observed in the presence of 5 × 10-6 M of chelerythrine; ANOVA with repeated measures; n = 5. ET-1 alone increased perfusion pressure by <6 mmHg, and the new baseline was used for calculation of increase in perfusion pressure.

In a separate study, it was observed that the concentration-dependent increase in skin perfusion pressure elicited by U-46619 was significantly (P < 0.05) higher in the presence of a low concentration of ET-1 (8 × 10-10 M) or the PKC activator PDBu (10-7 M) compared with U-46619 alone (Fig. 5). PDBu alone increased the baseline perfusion by 6-7 mmHg, and this was taken into consideration in the calculation of the amplification effect in perfusion pressure by PDBu.


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  		Fig. 5.   Effect of the PKC activator phorbol 12,13-dibutyrate (PDBu) on skin vasoconstrictor effect of U-46619. The concentration-dependent increase in perfusion pressure elicited by U-46619 was significantly (P < 0.05) higher in the presence of a low concentration of ET-1 (8 × 10-10 M) or PDBu (10-7 M) compared with U-46619 alone; ANOVA with repeated measures; n = 5. ET-1 alone increased perfusion pressure by <6 mmHg, and the new baseline was used for calculation of increase in perfusion pressure.

Role of Ca2+ in the Amplification Effect of a Low Concentration of ET-1 on U-46619-Induced Skin Vasoconstriction

The increase in skin perfusion pressure elicited by 5 × 10-7 M of U-46619 was enhanced by Ca2+ in the perfusion buffer in a concentration-dependent (10-5-10-2 M) manner (Fig. 6). The sensitivity to Ca2+ in the enhancement of U-46619-induced increase in skin perfusion pressure was significantly (P < 0.05) higher in the presence of a low concentration of ET-1 (8 × 10-10 M).


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  		Fig. 6.   Role of Ca2+ in the amplification effect of a low concentration of ET-1 on U-46619-induced (5 × 10-7 M) skin vasoconstriction. The sensitivity to Ca2+ in U-46619-induced increase in skin perfusion was significantly increased in the presence of a low concentration of ET-1 (8 × 10-10 M); ANOVA with repeated measures; n = 4, P < 0.05.

Role of Endogenous Cyclooxygenase Products in the Amplification of U-46619-Induced Vasoconstriction by a Low Concentration of ET-1

U-46619 elicited a concentration-dependent increase in skin perfusion pressure in the presence of 5 × 10-6 M indomethacin and L-NAME (Fig. 7). U-46619-induced increase in skin perfusion pressure was also significantly (P < 0.05) enhanced by pretreatment with a low concentration of ET-1 (8 × 10-10 M), even in the presence of 5 × 10-6 M of indomethacin and L-NAME. Infusion of indomethacin and L-NAME increased the baseline perfusion pressure by 9-10 mmHg, and this new baseline was used for the present study.


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  		Fig. 7.   Role of cyclooxygenase products in the amplification effect of a low concentration of ET-1 in U-46619-induced skin vasoconstriction. A low concentration of ET-1 (8 × 10-10 M) significantly (P < 0.05) potentiated the increase in skin perfusion pressure induced by U-46619 even in the presence of 5 × 10-6 M of indomethacin and Nomega -nitro-L-arginine methyl ester; ANOVA with repeated measures; n = 5. ET-1 alone increased perfusion pressure by <6 mmHg, and the new baseline was used for calculation of increase in perfusion pressure.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Major Findings in the Present Studies

Using the isolated perfused pig skin flap model, we previously demonstrated that pro-endothelin (big ET-1) is converted to vasoactive ET-1 in pig skin vasculature, and ET-1 is a potent and long-acting vasoconstrictor in pig skin (31, 32). Here, we further demonstrated that a low concentration of ET-1 may also contribute to the pathogenesis of skin vasospasm by potentiating the vasoconstrictor effects of other endogenous vasoactive substances. Specifically, we observed that a low concentration of ET-1 amplified the vasoconstrictor effect of NE in pig skin. This finding extended the observation reported by other investigators that subthreshold, threshold, or low concentrations of ET-1 enhanced the vasoconstrictor effect of NE in human and rabbit vascular rings (15, 42). More importantly, using the same skin flap model, we observed for the first time that a low concentration of ET-1 also amplified the skin vasoconstrictor effect of the TxA2 mimetic U-46619 independent of endogenous cyclooxygenase products, and the amplification mechanism probably involved activation of PKC and increase in sensitivity of the contractile apparatus to extracellular Ca2+ in smooth muscle cells.

Possible Mechanism for Amplification of U-46619-Induced Skin Vasoconstriction by a Low Concentration of ET-1

In the present studies, we obtained evidence to indicate that amplification of the vasoconstrictor effect of U-46619 by a low concentration of ET-1 is mediated by activation of intracellular PKC. This interpretation is based on the observation that the PKC inhibitor chelerythrine completely blocked the amplification effect of ET-1 on U-46619-induced skin vasoconstriction (Fig. 4). Conversely, a low concentration of the PKC activator PDBu potentiated the vasoconstrictor effect of U-46619 (Fig. 5). Of interest was the observation that the skin vasoconstrictor effect of U-46619 increased with increase in Ca2+ concentration in the perfusion buffer, and this Ca2+ concentration-dependent increase in skin vasoconstriction was potentiated in the presence of a low concentration of ET-1 (Fig. 6). Taken together, these observations led us to speculate that the amplification effect of ET-1 on U-46619-induced skin vasoconstriction probably involved activation of PKC, which in turn increased the sensitivity of the contractile apparatus of vascular smooth muscle cells to extracellular Ca2+.

Other investigators have studied the amplification mechanism of ET-1 on NE- or 5-HT-induced vasoconstriction in vascular rings. Specifically, Yang et al. (42) reported that ET-1 potentiated the vasoconstrictor effect of NE in human internal mammary rings by sensitizing the vascular smooth muscle to Ca2+. Subsequently, it was demonstrated in the rabbit aorta rings that sensitization of contractile apparatus to Ca2+ in vascular smooth muscle cells probably is the result of activation of PKC by ET-1 in amplification of NE-induced vasoconstriction (15). A similar mechanism was also reported for human umbilical artery in the potentiation of 5-HT-induced vasoconstriction by ET-1 (28). On the other hand, it was also reported that ET-1 potentiated the vasoconstrictor effect of 5-HT in rat mesenteric artery via release of TxA2 by nonendothelial cells (14). However, it is unlikely that endogenous TxA2 may play an important role in the amplification of U-46619-induced skin vasoconstriction in the present study because the cyclooxygenase inhibitor indomethacin did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction in the present study (Fig. 7).

In summary, we demonstrated that a low concentration of ET-1 potentiates the vasoconstrictor effect of NE and U-46619 in isolated perfused pig skin. The amplification mechanism of ET-1 on U-46619-induced skin vasoconstriction is independent of endogenous cyclooxygenase products. The amplification mechanism probably involves activation of PKC and sensitization of vascular smooth muscle cells to extracellular Ca2+.

Perspectives

The amplification effect of ET-1 on vasoconstrictor effects of other vasoactive substances such as NE and TxA2 observed in the present studies may have important clinical implication in the pathogenesis of skin vasospasm in trauma, skin ischemic disease, and skin flap surgery. Synthesis and release of ET-1 by endothelial cells are known to be stimulated by thrombin, NE, transforming growth factor-beta (TGF-beta ), and other substances (8, 12, 24, 25). Indeed, elevated circulating plasma levels of ET-1 have been reported in patients with skin ischemic disease (8, 12, 24), and elevated skin tissue and skin flap venous plasma level of ET-1 have been reported in laboratory animals undergoing skin flap surgery (22, 37). At sites where platelets are activated by trauma and/or damaged blood vessel walls, thrombin, which is formed after activation of the coagulation cascade by activated platelets, and TGF-beta and NE released from activated platelets will stimulate the synthesis and release of ET-1 in endothelial cells. In addition, NE is also released by traumatized sympathetic nerve endings, and NE, TxA2, and 5-HT are released by activated platelets. The vasoconstrictor effects of these vasoactive substances are most likely amplified by ET-1 in these sites. Therefore, the information obtained from the present studies provides important insight into the pathophysiology and pharmacologic intervention of skin vasospasm in trauma, skin ischemic disease, and skin flap surgery.


    ACKNOWLEDGEMENTS

The authors thank Tina Ferri for typing this manuscript.


    FOOTNOTES

This project was supported by operating Grant MT-8048 from the Medical Research Council of Canada to C. Y. Pang.

Address for reprint requests and other correspondence: C. Y. Pang, The Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario M5G 1X8, Canada (E-mail: pang{at}sickkids.on.ca).

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.

Received 24 July 2000; accepted in final form 16 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Afridi, NS, LiPaletz J, and Morris SF. Free flap failure: what to do next? Can J Plast Surg 8: 30-32, 2000.

2.   Angel, MF, Knight KR, Mellow CG, Morrison WA, and O'Brien BM. The effect of prior elevation of skin flaps and ischemia on blood thromboxane levels. Ann Plast Surg 22: 501-504, 1989[Web of Science][Medline].

3.   Anelini, GD, Christie MI, Bryan AJ, and Lewis MJ. Surgical preparation impairs release of endothelium derived factor from human saphenous vein. Ann Thorac Surg 48: 417-420, 1989[Abstract].

4.   Battistini, B, D'Orleans-Juste P, and Siroirs P. Biology of disease. Endothelins: circulating plasma levels and presence in other biological fluids. Lab Invest 68: 600-628, 1993[Web of Science][Medline].

5.   Consigny, PM. Endothelin-1 increases arterial sensitivity to 5-hydroxytryptamine. Eur J Pharmacol 186: 239-245, 1990[Web of Science][Medline].

6.   Daniel, RK, and Kerrigan CL. The omnipotential buttock skin flap. Plast Reconstr Surg 70: 11-16, 1982[Web of Science][Medline].

7.   Daniel, RK, and Kerrigan CL. Principles and physiology of skin flap surgery. In: Plastic Surgery, edited by McCarthy JG.. Philadelphia, PA: Saunders, 1990, vol. 1, p. 225-378.

8.   Doherty, AM. Endothelin structure and development of receptor antagonists. In: Chemical and Structural Approaches to Rational Drug Design, edited by Weiner DB, and William B.. Boca Raton, FL: CRC, 1995, p. 85-123.

9.   Edstrom, LF, Balkovich M, and Slotman GJ. Effect of ischemic skin flap elevation on tissue and plasma thromboxane A2, and prostacyclin production: modification by thromboxane synthesis inhibitor. Ann Plast Surg 20: 106-111, 1988[Web of Science][Medline].

10.   Forrest, CR, Xu N, and Pang CY. Evidence for nicotine-induced skin flap ischemic necrosis in the pig. Can J Physiol Pharmacol 72: 30-38, 1993.

11.   Gude, NM, King RG, and Brennecke SP. Autacoid interactions in the regulation of blood flow in the human placenta. Clin Exp Pharmacol Physiol 25: 706-711, 1998[Web of Science][Medline].

12.   Haynes, WG, and Webb DJ. Endothelin as a regulation of cardiovascular function in health and disease. J Hypertens 16: 1081-1098, 1998[Web of Science][Medline].

13.   He, W, Neligan P, Lipa J, Forrest C, and Pang CY. Comparison of secondary ischemic tolerance between pedicled and free island buttock skin flaps in the pig. Plast Reconstr Surg 100: 72-81, 1997[Web of Science][Medline].

14.   Hempelmann, RG, Pradel RHE, Mehdorn HM, and Ziegler A. Threshold concentrations of endothelin-1: effects on contractions induced by 5-hydroxytryptamine in isolated rat cerebral and mesenteric arteries. Pharmacol Toxicol 85: 115-122, 1999[Web of Science][Medline].

15.   Henrion, D, and Laher I. Potentiation of norepinephrine-induced contractions by endothelin-1 in the rabbit aorta. Hypertension 22: 78-83, 1993[Abstract/Free Full Text].

16.   Inoue, A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, and Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 86: 2863-2867, 1989[Abstract/Free Full Text].

17.   Inoue, A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, and Masaki T. The human prepoendothelin-1 gene. Complete nucleotide sequence and regulation of expression. J Biochem 264: 14954-14959, 1989.

18.   Inoue, H, Aihara M, Tomioka M, and Ishida H. Changes in endothelin-1, 6-keto-PGF1alpha and TxB2 in random pattern flaps. J Cardiovasc Pharmacol 31, Suppl.: S477-S479, 1998.

19.   Lewis, MJ, and Smith JA. Platelets, thrombosis, and the endothelium. In: Cardiovascular Significance of Endothelium-Derived Vasoactive Factors, edited by Rubanyi GM.. Mount Kisco, NY: Futura, 1991, p. 293-306.

20.   Lipa, JE, Neligan PC, Perreault TM, Baribeau J, Levine RH, Knowlton RJ, and Pang CY. Vasoconstrictor effect of endothelin-1 in human skin: role of ETA and ETB receptors. Am J Physiol Heart Circ Physiol 276: H359-H367, 1999[Abstract/Free Full Text].

21.   Lüsher, TF. Do we need endothelin antagonists. Cardiovasc Res 27: 2989-2093, 1993.

22.   Matzuraki, K. Effect of skin flap ischemia on plasma endothelin-1 levels. Ann Plast Surg 31: 499-503, 1993[Web of Science][Medline].

23.   May, JW, Jr, Chait LA, O'Brien BM, and Hurley JV. The no-reflow phenomenon in experimental free flaps. Plast Reconstr Surg 61: 256-267, 1978[Web of Science][Medline].

24.   Miller, RC, Pelton JT, and Huggins JP. Endothelins from receptors to medicine. Trends Physiol Sci 14: 54-60, 1993.

25.   Miyauchi, T. Pathophysiology of endothelin in the cardiovascular system. Annu Rev Physiol 61: 391-415, 1999[Web of Science][Medline].

26.   Monteiro-Riviere, K, Bowan F, Scheidt VJ, and Riviere JE. The isolated perfused porcine skin flap (IPPSF). II. Ultrastructure and histological characterization of epidermal viability. In Vitro Toxicol 1: 241-252, 1987.

27.   Nakayama, K, Ishigai Y, Uchida H, and Tanaka Y. Potentiation by endothelin-1 of 5-hydroxytryptamine-induced contraction in coronary artery of the pig. Br J Pharmacol 104: 978-986, 1991[Web of Science][Medline].

28.   Okatani, Y, Taniguchi K, and Sagara Y. Amplification effect of endothelin-1 on serotonin-induced vasoconstriction of human umbilical artery. Am J Obstet Gynecol 172: 1240-1245, 1995[Web of Science][Medline].

29.   Pang, CY, Chiu C, Zhong A, and Xu N. Pharmacologic intervention of skin vasospasm and ischemic necrosis in pigs. J Cardiovasc Pharmacol 21: 163-171, 1993[Web of Science][Medline].

30.   Pang, CY, Forrest CR, and Morris SF. Pharmacologic augmentation of skin flap viability: a hypothesis to mimic the surgical delay phenomenon or a wishful thought. Ann Plast Surg 22: 293-306, 1989[Web of Science][Medline].

31.   Pang, CY, Yang RZ, Neligan P, Xu N, Chiu C, Zhong A, and Forrest CR. Vascular effects and mechanism of action of endothelin-1 in isolated perfused pig skin. J Appl Physiol 79: 2106-2113, 1995[Abstract/Free Full Text].

32.   Pang, CY, Zhang J, Xu H, Lipa JE, Forrest CR, and Neligan PC. Role and mechanism of endothelin-B receptors in mediating ET-1-induced vasoconstriction in pig skin. Am J Physiol Regulatory Integrative Comp Physiol 275: R1066-R1074, 1998[Abstract/Free Full Text].

33.   Randall, MD, Edwards DH, and Griffin TM. Activities of endothelin-1 in the vascular network of the rabbit ear: a microangiographic study. Br J Pharmacol 101: 781-788, 1990[Web of Science][Medline].

34.   Riviere, JE. Isolated perfused porcine skin flap system. Pharm Biotechnol 8: 387-407, 1996[Medline].

35.   Riviere, JE, Bowman KF, Monteiro-Riviere NA, Dix LP, and Carver MP. The isolated perfused porcine skin flap (IPPSF). 1. A novel model for percutaneous absorption and cutaneous toxicology studies. Fundam Appl Toxicol 7: 444-453, 1986[Web of Science][Medline].

36.   Rogers, RA, and Riviere JE. Pharmacologic modulation of the cutaneous vasculature in the isolated perfused porcine skin flap. J Pharm Sci 83: 1682-1689, 1994[Web of Science][Medline].

37.   Tane, N, Inoue H, Aihara M, and Ishida H. The affects of endothelin-1 on flap necrosis. Ann Plast Surg 35: 389-395, 1995[Web of Science][Medline].

38.   Thomson, JG, and Kerrigan CL. Dermofluorometry: thresholds for predicting flap survival. Plast Reconstr Surg 83: 859-864, 1989[Web of Science][Medline].

39.   Wagner, OF, Christ G, Vierhapper H, Parzer S, Nowotny PJ, Schneider B, and Waldhause W. Polar secretion of endothelin-1 by culture endothelial cells. J Biol Chem 267: 16066-16068, 1992[Abstract/Free Full Text].

40.   Weinzweig, N, and Gonzalez M. Free tissue failure is not an all-or-none phenomenon. Plast Reconstr Surg 96: 648-660, 1995[Web of Science][Medline].

41.   Yanagisawa, M, Kurihawa H, Kimur S, Tombe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Sasaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1998.

42.   Yang, Z, Richard V, von Segesser L, Bauer E, Stulz P, Turina M, and Lüscher TF. Threshold concentrations to norepinephrine and serotonin in human arteries. A new mechanism of vasospasm? Circulation 82: 188-195, 1990[Abstract/Free Full Text].

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