Mechanisms of Altered Contractile Responses to Vasopressin and Endothelin in Canine Coronary Collateral Arteries
Background Mature coronary collateral arteries are hyperresponsive to vasopressin; in contrast, contractile responses of collaterals to endothelin are attenuated. Our goal was to determine the cellular mechanisms underlying these differences in reactivity using two sizes of canine collateral arteries isolated from hearts subjected to chronic coronary occlusion.
Methods and Results Contractile responses to vasopressin (100 nmol/L) were enhanced threefold to fourfold in near-resistance (≈200 μm lumen diameter) and conduit (≈500 μm lumen diameter) collateral arteries compared with similarly sized noncollateral coronary arteries (P<.01). In contrast, contractions of both sizes of collaterals in response to endothelin (0.01 to 30 nmol/L) were smaller than responses of size-matched noncollateral arteries (P<.05). Pretreatment with either indomethacin (5 μmol/L), a cyclooxygenase inhibitor, or NG-nitro-l-arginine methyl ester (100 μmol/L), a nitric oxide synthase inhibitor, did not alter the relative responsiveness of collateral arteries to vasopressin or endothelin compared with noncollateral arteries. Vasopressin produced greater increases of intracellular free Ca2+ (measured by use of fura-2 microfluorometry and Ca2+-dependent 42K+ efflux) in smooth muscle of collateral arteries than in smooth muscle of noncollateral arteries (P<.05). Surprisingly, endothelin-induced increases of Ca2+ were not different in smooth muscle of collateral and noncollateral arteries (P>.05).
Conclusions We conclude that altered contractile responsiveness of collateral arteries to vasopressin and endothelin does not result from altered synthesis/release of nitric oxide or prostaglandins. Parallel enhancement of vasopressin-mediated Ca2+ and contractile responses suggests increases in vasopressin receptor number, affinity, and/or efficiency of coupling mechanisms in collateral smooth muscle. The dissociation between endothelin-induced contractile and Ca2+ responses of collaterals indicates that the mechanisms involved in increasing Ca2+ sensitivity of contractile proteins during endothelin stimulation may be altered in collateral arteries.
The coronary collateral circulation is capable of remarkable growth and development in response to gradual coronary occlusion.1 2 3 Blood flow through well-developed coronary collateral arteries is sufficient to maintain normal resting perfusion to myocardium located distal to a coronary occlusion.1 4 5 6 In vivo and in vitro studies7 8 9 10 have determined that collateral arteries respond to numerous vasoactive agonists and thus play an active role in regulation of blood flow to myocardium distal to an occlusion. However, the contractile responsiveness of collateral arteries differs from that of similarly sized normal coronary arteries. Specifically, contractile responses of canine coronary collateral arteries to vasopressin have been shown to be markedly enhanced compared with normal coronary arteries both in vivo11 and in vitro.7 In contrast, our laboratory12 and others10 have demonstrated that collaterals are significantly less responsive to endothelin than are similarly sized normal coronary arteries. Collateral arteries have also been reported to exhibit attenuated contractile responses to prostaglandin F2α,13 K+,10 13 and the thromboxane mimetic U46619.10
The purpose of our study was to evaluate cellular mechanisms underlying the altered contractile responsiveness of coronary collateral arteries. We studied the differential reactivity of collaterals to the endogenous vasoconstrictors vasopressin and endothelin. Because agonist-mediated vasoconstriction is often modulated by simultaneous synthesis/release of vasoactive factors by the endothelium, our first objective was to determine whether the altered contractile responses of collaterals to endothelin and vasopressin resulted from changes in production of either nitric oxide or prostaglandins by the endothelium. Both vasopressin and endothelin have been shown to stimulate the release of these substances.14 15 16 17 Altman and Bache18 reported increased basal production of prostaglandins in collateral arteries in vivo. Thus, differential effects of vasopressin or endothelin on production of endothelium-derived vasodilators in collateral arteries could conceivably account for the altered contractile responses to these agonists. To determine the roles of nitric oxide and prostaglandins, we compared the effects of inhibition of nitric oxide synthase or cyclooxygenase on contractions of collateral and noncollateral arteries in response to vasopressin and endothelin.
The second objective of our study was to evaluate cellular mechanisms in the smooth muscle of collateral arteries that potentially could be involved in the altered contractile responses of collateral arteries. We hypothesized that the altered contractile responses of collateral arteries to vasopressin and endothelin result from parallel changes in receptor-dependent intracellular Ca2+ regulation in the smooth muscle of these arteries. We used two techniques to compare intracellular Ca2+ responses to vasopressin and endothelin in the smooth muscle of collateral and noncollateral coronary arteries: (1) fura-2 microfluorometry and (2) measurements of Ca2+-dependent 42K efflux. The ratio of fura-2 fluorescences is a relative measure of average myoplasmic free Ca2+ concentration (Cam).19 42K efflux reflects the opening of large conductance Ca2+-dependent K+ channels and therefore is sensitive to the free Ca2+ concentration in the subsarcolemmal space (Cas). Differences between Ca2+ responses measured by fura-2 microfluorometry or 42K efflux could reveal heterogeneity in the myoplasmic distribution of Ca2+ in collateral and noncollateral arteries. We simultaneously measured changes in contractile tension and fura-2 fluorescence in response to vasopressin and endothelin to permit direct correlation between time-dependent changes in tension and Cam in response to these agonists. This study contains the first measurements of intracellular free Ca2+ in the smooth muscle of coronary collateral arteries.
Induction of Collateral Artery Growth
Adult mongrel male dogs (weight, ≈25 to 35 kg) were anesthetized with acepromazine maleate (0.8 mg/kg SC) and sodium pentobarbital (25 mg/kg IV) and ventilated mechanically. An aneroid constrictor (2.75 to 4.0 mm ID, Research Instruments and MFG) was placed around the proximal LCx coronary artery by use of sterile techniques. During surgery and recovery, dogs received buprenorphine hydrochloride (0.01 mg/kg IV or IM) as needed for pain relief. Antibiotics were given immediately before surgery (900 000 U penicillin IM) and for 5 days after surgery (800 mg sulfamethoxazole and 160 mg trimethoprim). All experimental procedures were in accordance with the “Position of the American Heart Association on Research Animal Use” adopted November 11, 1984, and were approved by the Animal Care and Use Committee of the University of Missouri.
Preparation of Coronary Artery Rings for Contraction Studies
Coronary arteries were isolated 4 months after implantation of the aneroid occluder. On the day of the experiment, the dogs were anesthetized with sodium pentobarbital (40 mg/kg), and their hearts were removed rapidly and placed in cold Krebs' bicarbonate buffer. Complete closure of the LCx artery was confirmed in all experiments. Collateral arteries were easily identified as tortuous, epicardial vessels extending between branches of the LAD artery and branches of the LCx artery. We isolated conduit (≈500 μm ID) and near-resistance–sized (≈200 μm ID) arteries. Size-matched epicardial noncollateral branches of the LAD (not exposed to chronic occlusion) and LCx (collateral-dependent) arteries were isolated from the same hearts.
Conduit Coronary Arteries
Conduit coronary arteries were cut into rings with axial lengths of 3.5 to 4.0 mm. Vessel dimensions (OD, ID, and vessel wall thickness) were measured with a Filar eyepiece micrometer (Hitschfel Instruments, Inc) using a thin ring cut from the end of each artery; rings were measured in a relaxed, nonpressurized state. Arteries were mounted on two stainless steel wires (Rocky Mountain Orthodontics); one was attached to a force transducer (model FT.03 c, Grass Instrument Co), and the other was attached to a micrometer (Stoelting/Prior Microdrive). Rings were lowered into 20-mL tissue baths containing Krebs' bicarbonate buffer (37°C). After 1 hour of equilibration, arteries were progressively stretched to the optimum of the length–active tension relationship, as indicated by contractile responses to 30 mmol/L K+ measured at each level of stretch. Optimal length (Lmax) was defined as the circumferential length at which the active tension produced was <5% greater than the tension produced at the previous length. We have found that length–active tension relationships are qualitatively similar in collateral, LAD, and LCx arteries. Lmax and resting tension at Lmax were not significantly different between the three vessel types. All protocols were performed with arteries stretched to Lmax.
Near-Resistance Coronary Arteries
Near-resistance coronary arteries were cut into rings with axial lengths of ≈1.5 mm and studied by use of microvessel myographs (Living Systems Instruments) as previously described.20 21 Dimensions of arterial rings were measured from a thin section cut from the end of each ring as described for conduit arteries. Each ring was threaded carefully onto two 20-μm tungsten wires; one was secured to a force transducer (Kulite Semiconductor Products Inc), and the other attached to a digital micrometer. Arteries were superfused with Krebs' bicarbonate buffer (37°C, 6 mL/min). After 45 to 60 minutes' equilibration, arteries were systematically stretched to Lmax. Length–passive tension and length–active tension relationships were determined with the use of repetitive stretches and K+-induced contractions. Vessels were maintained at Lmax throughout the experiments.
In Vitro Evaluation of Contractile Responses
Concentration-response relationships to endothelin were determined by cumulative additions of small aliquots of concentrated stock solutions directly to the tissue bath (conduit arteries) or recirculating perfusate (near-resistance arteries). We evaluated responses of coronary arteries to a single near-maximal concentration of vasopressin. Some rings were incubated continuously with L-NAME or indomethacin beginning at least 10 minutes before evaluation of contractile responses. For conduit arteries, we performed paired experiments with two rings cut from each artery and studied responses in the absence and presence of the selected inhibitor.
Single Smooth Muscle Cells
Agonist-mediated changes in fura-2 fluorescence were measured in single smooth muscle cells freshly dispersed from conduit collateral and noncollateral coronary arteries (≈500 μm ID). The methods for enzymatic dispersion of smooth muscle cells from coronary arteries have been described previously.22 Briefly, arteries were cut open longitudinally and pinned lumen side up in solution consisting of low-Ca2+ (0.5 mmol/L) HEPES-buffered PSS plus 294 U collagenase, 2 mg BSA, 1 mg soybean trypsin inhibitor, and 0.4 mg DNase I per milliliter. Smooth muscle cells were dispersed for 1 to 1.5 hours in a shaking water bath at 37°C. Cells were loaded with 2.5 μmol fura-2/AM (Molecular Probes) per liter for 20 minutes, rinsed for 30 minutes in sterile media, and resuspended in HEPES-buffered PSS containing 2% BSA.
Fura-2 measurements of Cam were obtained by use of microfluorometry methods described previously.23 Briefly, freshly dispersed smooth muscle cells were placed in a superfusion chamber mounted on the stage of an inverted microscope. Light from a Xenon arc lamp passed to the cells via a liquid light guide through a rotating wheel containing 340- and 380-nm interference filters. The fluorescence emission at 510 nm was defined spatially to the area of only one cell with an adjustable aperture and reflected to a photomultiplier tube with a dichroic mirror. Fluorescence was analyzed with an analog fluorescence signal processor and an analog-to-digital convertor.23 Subtraction of background fluorescence and calculation of ratio were performed on line by use of customized AxoBASIC 1.0 software (Axon Inc).
Simultaneous Measurement of Contractile Tension and Fura-2 Fluorescence
Contractile tension and Cam were measured simultaneously in near-resistance coronary rings by use of a specially designed microvessel myograph and microfluorometry methods described above.24 Measurement of fura-2 fluorescence in intact conduit collateral arterial rings was not feasible because of high autofluorescence. Smooth muscle of near-resistance arterial rings was loaded with fura-2 by incubation with fura-2/AM (10 μmol/L) for 2 hours at 37°C. The loading solution contained 0.5% cremophor el and 5% BSA. Arteries were rinsed for 30 minutes in media at 37°C to remove extracellular fura-2/AM. Changes in contractile tension were measured by use of techniques similar to those described above; all experiments were performed with arteries stretched to Lmax. Autofluorescence of the arterial rings was determined at the end of each experiment by quenching the Ca2+-sensitive fura-2 fluorescence via exposure of the arteries to 10 mmol/L Mn2+ and 5 μmol/L ionomycin.22 25 The fluorescence ratio was calculated after subtraction of autofluorescence.
42K Efflux Measurements
Measurements of 42K efflux were performed by use of methods described previously for other arteries.26 Strips of coronary arteries were mounted on stainless steel wire holders for measurements of ion flux. Tissues were incubated for 2 hours with 42K (20 μCi/mL; University of Missouri Research Reactor). This incubation period ensured complete equilibration of the cellular pools. Arteries were rinsed for 2 seconds in nonradioactive Krebs' solution to remove the surface isotope and then passed through a series of vigorously gassed tubes containing the experimental solutions. At the end of the experiment, tissues and loading solution standards were extracted into an acid solution and were counted by liquid scintillation techniques. Washout curves were derived by reverse-order sequential addition of the counts in tissues and tubes (corrected for background and isotope decay) followed by division of the counts remaining after each period by the counts at time zero. Rate of efflux was expressed as fractional loss of isotope per minute and was presented as the rate constant (k, min−1).
Solutions and Drugs
The Krebs' bicarbonate buffer used for all studies of contractile function contained (in mmol/L) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 25 NaHCO3, and 10.1 glucose (gassed with 95% O2 and 5% CO2, pH 7.4, 37°C). This solution also contained 3 μmol propranolol and 25 μmol EDTA per liter. The PSS used for superfusion of single smooth muscle cells contained (in mmol/L): 138 NaCl, 2 CaCl2, 5 KCl, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). Solutions used for depolarizing smooth muscle were produced by equimolar replacement of NaCl with KCl. Unless otherwise indicated, drugs were purchased from Sigma Chemical Co. We purchased endothelin-1 from Peninsula Laboratories, Inc and ionomycin from Calbiochem.
Contractions of conduit coronary rings were expressed as absolute values in grams of developed tension. Contractile tension produced by near-resistance coronary arteries was presented as the active force (in millinewtons) divided by the axial length of the vessel segment (in millimeters).21 Contractile responses were also presented as a percentage of the maximal K+-induced contraction. EC50 values were determined by use of nonlinear regression analysis of concentration-response data.
Measurements of Cam were expressed as the ratio of fura-2 fluorescence at 340- and 380-nm excitation wavelengths rather than absolute Ca2+ concentration because of uncertainties regarding extrapolation of in vitro calibrations to in vivo measurements.22 To determine whether the properties of fura-2 were the same in smooth muscle of collateral, LAD, and LCx arteries, we measured minimum ratio (Rmin) and maximum ratio (Rmax) in single smooth muscle cells and intact arterial rings from a subset of dogs. In single cells, Rmin was determined in HEPES-buffered PSS lacking extracellular Ca2+ and containing 2 mmol EGTA and 2 μmol ionomycin per liter. Rmax was measured by use of a solution containing 2 μmol ionomycin and 10 mmol Ca2+ per liter. For intact near-resistance arterial rings, the Rmin solution consisted of Krebs' solution without extracellular Ca2+ and with 2 mmol EGTA and 10 μmol ionomycin per liter. The solution used to determine Rmax in smooth muscle of intact arteries contained 5 mmol Ca2+ and 10 μmol ionomycin per liter. Rmin and Rmax values measured in near-resistance collateral, LAD, and LCx arteries and in smooth muscle cells from conduit collateral, LAD, and LCx arteries were not significantly different.
Concentration-response curves were compared by use of two-way ANOVA. When a significant F value was found, differences between groups were ascertained by use of Fisher's test for least significant difference. EC50 values and responses to single doses of agonists were compared by use of two-way ANOVA, and pairwise comparisons between groups were performed with the Student-Newman-Keuls test. Comparisons of proportions of cells from each artery type that responded to an agonist (percentage of responders) were performed by use of χ2 analysis. A value of P<.05 was considered significant. Data are presented as mean±SEM, and n values indicate the number of animals. Each animal was represented once per data point.
Dimensions of the two sizes of coronary arteries studied are presented in Table 1⇓. Conduit and near-resistance collateral arteries had thicker vessel walls than similarly sized noncollateral arteries (P<.05). IDs of conduit collateral arteries were slightly smaller than IDs of LAD and LCx arteries (P<.05), but ODs of the three artery types were not significantly different. IDs of near-resistance–sized arteries were not different (P>.05). However, the thicker walls of near-resistance collateral arteries resulted in these arteries having significantly larger ODs than respective LAD and LCx arteries (P<.05).
Depolarization with K+ produced significantly smaller contractions of collateral arteries compared with contractions of LAD and LCx arteries (P<.01). Contractions of conduit collateral, LAD, and LCx arteries in response to 100 mmol/L K+ averaged 4.9±0.7, 11.1±0.7, and 11.4±0.8 g, respectively (n=12). Contractions of near-resistance collateral, LAD, and LCx arteries in response to 80 mmol/L K+ averaged 3.7±0.5, 5.5±0.5, and 5.1±0.4 mN/mm, respectively (n=21).
Contractile Responses to Vasopressin and Endothelin
Contractions of conduit and near-resistance collateral arteries in response to vasopressin (100 nmol/L) were significantly larger than contractions of size-matched LAD and LCx arteries (P<.01; Fig 1⇓). In contrast, contractile responses of collaterals to endothelin were significantly attenuated compared with LAD and LCx arteries (P<.05; Fig 2⇓). In addition to diminished contractions, collateral arteries were less sensitive to endothelin; EC50 values for near-resistance collateral, LAD, and LCx arteries averaged 4.1±0.7, 1.8±0.2, and 2.0±0.3 nmol/L, respectively (P<.01).
Maximal endothelin-induced contractions of collateral arteries remained significantly attenuated compared with LAD and LCx arteries after normalization to maximal K+-induced contractions (P<.01). Maximal contractions in response to 30 nmol/L endothelin averaged 31±9%, 61±10%, and 57±8% of the maximal K+ response in conduit collateral, LAD, and LCx arteries, respectively. Maximal endothelin-induced contractions of near-resistance collateral, LAD, and LCx arteries were 40±13%, 115±12%, and 115±13% of the maximal K+ response.
Role of Nitric Oxide and Prostaglandins in Modulating Contractile Responses
We evaluated the effect of inhibition of either nitric oxide synthase or cyclooxygenase on contractile responses of conduit collateral and noncollateral arteries to 100 nmol/L vasopressin. Contractions of conduit collateral arteries in response to vasopressin remained significantly larger than responses of LAD and LCx arteries in the presence of either 100 μmol/L L-NAME or 5 μmol/L indomethacin (P<.05; Fig 3⇓). Results of paired experiments indicated vasopressin-induced contractions of conduit collateral, LAD, and LCx arteries were not significantly altered by pretreatment with L-NAME or indomethacin. Similarly, vasopressin-induced contractions of near-resistance collateral arteries remained significantly greater than contractions of near-resistance noncollateral arteries in the presence of L-NAME (collateral arteries, 2.27±0.57 mN/mm versus 0.10±0.05 for LAD arteries and 0.74±0.58 for LCx arteries; P<.05).
Neither L-NAME nor indomethacin altered the relative responsiveness of collateral arteries to endothelin. Contractions of conduit collateral arteries in response to 30 nmol/L endothelin remained significantly smaller than contractions of LAD and LCx arteries in the presence of either L-NAME or indomethacin (Fig 4⇓). Endothelin responses of all three arteries were not significantly altered by the presence of L-NAME. Pretreatment with indomethacin did not affect the responses of conduit collateral and LAD arteries to endothelin; however, contractions of conduit LCx arteries were enhanced by indomethacin (P<.05). Contractile responses of near-resistance collateral arteries remained decreased compared with LAD and LCx arteries in the presence of either L-NAME or indomethacin (P<.05; Fig 5⇓).
Ca2+ Responses to Vasopressin
Cam Responses in Near-Resistance Arteries: Simultaneous Measurement of Fura-2 Fluorescence and Contractile Tension
Vasopressin (100 nmol/L) produced biphasic increases of Cam in smooth muscle of collateral arteries and monophasic Cam increases in LAD and LCx arteries (Fig 6⇓, top). Increases of Cam in collateral smooth muscle were significantly greater than in LAD and LCx smooth muscle during the first 90 seconds of the response (P<.05). Contractile responses of all arteries to vasopressin were transient (Fig 6⇓, bottom). Vasopressin-induced contractile responses of collateral arteries were significantly larger than responses of noncollaterals (P<.05).
Cam Responses in Conduit Arteries: Fura-2 Microfluorometry in Single Smooth Muscle Cells
Vasopressin-induced Cam responses of conduit collateral arteries were evaluated by use of single smooth muscle cells freshly dispersed from these arteries. We observed Cam responses to vasopressin in collateral smooth muscle cells from 82% of dogs, whereas fewer than 10% of dogs had cells from LCx and LAD arteries that responded (Table 2⇓). The percentage of dogs with collateral smooth muscle cells that responded to vasopressin was significantly larger than the percentages of dogs with LCx and LAD cells that responded (P<.05). Average changes in Cam in collateral and LCx cells that responded to vasopressin are illustrated in Fig 7⇓.
Cas Responses in Conduit and Near-Resistance Arteries: Measurement of 42K Efflux
Measurement of 42K efflux was used to evaluate changes in Cas in response to vasopressin in collateral, LAD, and LCx coronary arteries.19 26 Vasopressin-induced increases of 42K efflux from smooth muscle of conduit and near-resistance collateral arteries were significantly larger than increases produced in smooth muscle of size-matched LAD and LCx arteries (P<.01; Fig 8⇓). The vasopressin1a-selective receptor antagonist [β-mercapto-β,β-cyclopentamethylenepropionyl1,o-methyltyrosine2] arginine vasopressin abolished vasopressin-induced increases in 42K efflux in conduit collateral arteries (data not shown, n=4).
Ca2+ Responses to Endothelin
Cam Responses in Near-Resistance Arteries: Simultaneous Measurement of Fura-2 Fluorescence and Contractile Tension
Endothelin produced concentration-dependent increases of Cam in collateral and noncollateral arteries up to the concentration of 3.0 nmol/L (Fig 9⇓, top). At higher concentrations (10 and 30 nmol/L), Cam did not increase further, but contractile tension produced by all three arteries continued to increase in response to increasing concentrations of endothelin (Fig 9⇓, bottom). Contractile responses of near-resistance collateral arteries to endothelin were significantly smaller than responses of LAD and LCx arteries. In contrast, endothelin-induced Cam responses in collateral arteries were not attenuated. Peak and plateau Cam responses were not significantly different in collateral, LAD, and LCx arteries (P>.05).
Cam Responses in Conduit Arteries: Fura-2 Microfluorometry in Single Smooth Muscle Cells
As explained previously, Cam responses were measured in single smooth muscle cells freshly dispersed from conduit collateral and noncollateral arteries. Endothelin (100 nmol/L) produced indistinguishable increases of Cam in smooth muscle cells from conduit collateral, LAD, and LCx coronary arteries (P>.05, Fig 10⇓).
Cas Responses in Conduit and Near-Resistance Arteries: Measurement of 42K Efflux
We measured changes in 42K efflux in response to increasing concentrations of endothelin (0.3 to 100 nmol/L) in conduit and near-resistance collateral and noncollateral coronary arteries. Endothelin significantly increased 42K efflux in all arteries in a concentration-dependent manner. The increases were not significantly different between the three artery types (P>.05; data not shown).
The goal of the present study was to characterize the altered contractile responsiveness of coronary collateral arteries to endothelin and vasopressin and to determine potential mechanisms underlying the altered responses. We determined that two sizes of collateral arteries (≈500 and ≈200 μm ID) are hyperresponsive to vasopressin. Furthermore, we demonstrated that these collateral arteries exhibit reduced contractile responsiveness to endothelin. We found that the altered contractile responsiveness of collaterals to these potent endogenous vasoconstrictors does not appear to result from altered production of nitric oxide or prostaglandins in response to these agonists.
This study is the first investigation of cellular mechanisms underlying the altered contractile responsiveness of coronary collateral smooth muscle. We studied vasopressin- and endothelin-induced changes in Cam using the fluorescent Ca2+ indicator fura-2 and changes in Cas using measures of 42K efflux. Our evaluation of vasopressin- and endothelin-induced increases of Cam and Cas provides insight into mechanisms potentially involved in the altered contractile responses of collaterals to these vasoconstrictors. Parallel increases in vasopressin-mediated increases of Cam, Cas, and contractile tension suggest alterations in vasopressin receptor number, affinity, and/or coupling mechanisms in smooth muscle of collateral arteries. In contrast to findings with vasopressin, contractile responses of collaterals to endothelin are attenuated whereas increases of Cam and Cas do not differ from those of noncollateral arteries. These results suggest an altered relationship between Ca2+ and contractile tension (decreased Ca2+ sensitivity of contractile proteins) during endothelin stimulation of collateral artery smooth muscle.
Role of Nitric Oxide and Prostaglandins
In addition to direct contractile effects on vascular smooth muscle, vasopressin and endothelin have been shown to stimulate production of nitric oxide27 28 and prostaglandins.15 17 29 30 The effects of vasopressin and endothelin on activity of nitric oxide synthase and cyclooxygenase have been shown to vary considerably with vessel type. For example, vasopressin induces relaxation of canine basilar arteries that is endothelium dependent and mediated by nitric oxide.16 31 In contrast, vasopressin produces relaxation of large canine coronary arteries that is only partially dependent on the endothelium31 32 33 and produces contraction of small conduit coronary branches7 and coronary microvessels.33 34 Vasopressin-induced constriction of coronary microvessels appears to be limited by simultaneous stimulation of synthesis/release of nitric oxide.33 Results of studies in various species and vasculature types indicate that endothelin may stimulate synthesis of nitric oxide,15 35 prostaglandins,15 or both.36 Endothelin-induced vasoconstriction has been shown to be enhanced by indomethacin,15 37 hemoglobin,15 methylene blue,15 or inhibitors of nitric oxide synthesis.28 These observations indicate that concomitant production of nitric oxide and prostaglandins limits the vasoconstrictor activity of endothelin.
Thus, altered synthesis of either nitric oxide or prostaglandins in response to vasopressin and endothelin could conceivably account for the altered contractile responsiveness of coronary collateral arteries. In this regard, Sellke and colleagues38 observed significant enhancement of vasopressin-induced contractile responses of collateral-dependent coronary microvessels. However, in the presence of hemoglobin, which inactivates nitric oxide, the contractions of control and collateral-dependent microvessels in response to vasopressin were not significantly different. Therefore, these authors concluded that the primary reason for enhanced vasopressin-induced constriction of collateral-dependent coronary microvessels involved decreased release of nitric oxide.
In contrast, our results do not implicate altered production of nitric oxide or prostaglandins as the mechanism of enhanced contractile responsiveness of coronary collateral arteries to vasopressin. Neither inhibition of nitric oxide synthesis with L-NAME nor inhibition of synthesis of prostaglandins with indomethacin changed the relative responsiveness of collateral arteries to endothelin and vasopressin compared with noncollateral arteries (Figs 3 and 5⇑⇑). Our findings are consistent with the preliminary report of Altman and Dulas,39 who also observed that contractions of canine coronary collateral arteries in response to endothelin remained significantly diminished in the presence of L-NAME and indomethacin. Therefore, altered contractile responses of collateral arteries to endothelin and vasopressin do not appear to result from differences in the production of nitric oxide or prostaglandins. Alternatively, our data indicate that the altered contractile responsiveness of collaterals is likely due to changes in the mechanisms mediating the contraction of collateral artery smooth muscle.
Although production of nitric oxide and prostaglandins does not appear to be altered in collateral arteries, our results raise the possibility of increased production of vasodilator prostaglandins in collateral-dependent arteries. Although indomethacin did not alter contractions of collateral and LAD arteries, pretreatment with this inhibitor significantly enhanced endothelin-induced contractions of LCx arteries. Furthermore, vasopressin-induced contractions of LCx arteries also tended to be enhanced by indomethacin (P=.07). These results suggest that collateral-dependent arteries may tonically release vasodilator prostaglandins. Although this effect of indomethacin has not been previously described in collateral-dependent arteries, Altman and Bache18 determined that cyclooxygenase inhibition decreased collateral blood flow in vivo, as determined by retrograde blood flow measurements.
Mechanisms of Enhanced Responsiveness of Collateral Smooth Muscle to Vasopressin
Vasopressin-induced increases of Ca2+ in smooth muscle of collateral arteries appear to be increased in parallel to the enhanced contractile responsiveness. Vasopressin increased Ca2+-dependent 42K efflux in collateral artery smooth muscle threefold to fourfold more than in smooth muscle of noncollateral coronary arteries (Fig 8⇑). This finding indicates that vasopressin produces markedly greater increases of Cas in collateral smooth muscle than in normal coronary smooth muscle. Using fura-2 microfluorometry, we observed greater increases of Cam in near-resistance collateral arteries than in LAD and LCx arteries (Fig 6⇑). Similarly, in single smooth muscle cells freshly dispersed from conduit collateral arteries, we observed vasopressin-mediated Cam responses that were rarely observed in smooth muscle cells from conduit LCx arteries and were never observed in cells dispersed from LAD arteries. Taken in concert, all of our data are consistent with our hypothesis that vasopressin-induced increases of Ca2+ in collateral artery smooth muscle are greater than increases observed in noncollateral coronary smooth muscle. The parallel enhancement of Cam, Cas, and contractile responses to vasopressin in collateral arteries suggests that the alteration in collateral artery smooth muscle involves cellular mechanisms that precede the generation of a rise in intracellular Ca2+. A likely possibility is that smooth muscle of collateral arteries contains more vasopressin receptors than smooth muscle of normal coronary arteries. However, changes in vasopressin receptor affinity and/or the ability of receptors to couple to intracellular signaling mechanisms could also account for the hyperresponsiveness.
Mechanisms of Decreased Responsiveness of Collateral Smooth Muscle to Endothelin
In contrast to our findings with vasopressin, we were surprised to find that endothelin-induced Ca2+ responses in collateral artery smooth muscle were not decreased in parallel with the attenuated contractile responses. Increases of Cam (fura-2 fluorescence ratio) and increases of Cas (Ca2+-dependent 42K efflux) in response to endothelin were not significantly different in smooth muscle of conduit and near-resistance collateral, LAD, and LCx coronary arteries. The similar Ca2+ responses observed in collateral and noncollateral artery smooth muscle suggest that the receptors and signaling mechanisms responsible for endothelin-induced increases of Ca2+ are comparable in smooth muscle of collateral and noncollateral arteries. Instead, the dissociation between intracellular free Ca2+ concentration and contractile tension during endothelin stimulation suggests that mechanisms involved in increasing Ca2+ sensitivity of contractile proteins during endothelin stimulation may be altered in collateral artery smooth muscle. Endothelin, like many vasoconstrictors, has been shown to increase the Ca2+ sensitivity of contractile proteins.40 41 The Ca2+-sensitization effects of endothelin have been shown to be mediated by G protein(s) and potentially by activation of protein kinase C.40 The latter conclusion was based on inhibition of these effects by staurosporine and chelerythrine, somewhat nonselective inhibitors of protein kinase C. However, Shimamoto and colleagues42 observed that protein kinase C inhibition with a more selective inhibitor, calphostin C, inhibited the endothelin-induced contractile response of rat aorta by only 13.2% and 25.8% in the presence and absence of extracellular Ca2+, respectively. These results suggest that the contribution of protein kinase C activity to endothelin-induced vasoconstriction is relatively small. Furthermore, Hori and coworkers43 observed that the Ca2+ sensitization effects of endothelin were independent of protein kinase C activity. Thus, increases of Ca2+ sensitivity of contractile proteins in vascular smooth muscle in response to endothelin may be due to mechanisms dependent and independent of activation of protein kinase C. Alterations in one or both types of these mechanisms in collateral artery smooth muscle would explain the disparity we observed in Ca2+ and contractile responses to endothelin in collateral arteries. However, additional studies are required to confirm whether mechanisms involved in endothelin-induced Ca2+ sensitization are altered in collateral artery smooth muscle.
Our results indicate that differential alterations in reactivity exist in smooth muscle of conduit and near-resistance coronary collateral arteries to vasopressin and endothelin. Collateral arteries exhibit markedly enhanced contractile responsiveness to vasopressin while exhibiting significantly attenuated responsiveness to endothelin. Our evaluation of intracellular Ca2+ responses in collateral smooth muscle has provided insight into the cellular mechanisms underlying these changes in reactivity. The hyperresponsiveness of collateral artery smooth muscle to vasopressin may involve increases in number or affinity of vasopressin receptors or in the efficiency of receptor coupling. In contrast, the attenuated responsiveness of collateral arteries to endothelin does not appear to involve a decrease in endothelin receptor number or affinity. Instead, our data suggest that mechanisms involved in endothelin-induced Ca2+ sensitization of myofilaments may be altered in smooth muscle of collateral arteries.
Selected Abbreviations and Acronyms
|Cam||=||average myoplasmic free Ca2+ concentration|
|Cas||=||free Ca2+ concentration in the subsarcolemmal space|
|fura-2/AM||=||fura-2 acetoxymethyl ester|
|LAD||=||left anterior descending|
|Lmax||=||optimal artery circumferential length|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|PSS||=||physiological saline solution|
These studies were supported by research funds from the American Heart Association and the National Institutes of Health: HL-47812, training grant HL-07094, and program project PO1 HL-52490. Dr Sturek is the recipient of Research Career Development Award HL-02872. Dr Rapps was supported by a predoctoral fellowship from the American Heart Association, Missouri Affiliate. The authors greatly appreciate the technical contributions made by M.L. Mattox, D. Wycoff, and Q. Zhong.
Reprint requests to J.L. Parker, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211. E-mail janet_ firstname.lastname@example.org.
- Received March 26, 1996.
- Revision received August 8, 1996.
- Accepted August 19, 1996.
- Copyright © 1997 by American Heart Association
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