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Am J Physiol Heart Circ Physiol 280: H1019-H1028, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 3, H1019-H1028, March 2001

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries

Surya M. Nauli, Lubo Zhang, and William J. Pearce

Departments of Physiology and Pharmacology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC50) = 4.4 ± 0.1 M] than in adult (-log EC50 = 4.0 ± 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC50 = 4.9 ± 0.1 M) than in adult (-log EC50 = 4.7 ± 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 µM significantly reduced calcium only in fetal basilars (Delta  = 33 ± 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta  = 29 ± 6 and Delta  = 42 ± 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta -escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 µM, respectively) than in adult (410 and 231 µM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.

8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate; cerebral arteries; cerebrovascular circulation; guanylate cyclase; intracellular calcium concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

COMPARED WITH ADULTS, neonates are more vulnerable to a broad variety of cerebrovascular insults such as asphyxia and cerebral ischemia. Although this difference may reflect the greater water content and thinner wall thicknesses typical of immature cerebral arteries in human infants (21), functional immaturity of the mechanisms governing cerebrovascular contractility also undoubtedly plays a role. For example, fetal and adult cerebral arteries from both animals and humans exhibit different calcium sensitivities, different patterns of calcium mobilization, and different efficiencies of intracellular calcium buffering (4, 18). Compared with adult cerebral arteries, fetal cerebral arteries are generally more sensitive to agonists in multiple species, including baboons (14) and sheep (19). Fetal cerebral arteries in sheep are more dependent on calcium influx for activation (4, 33) and exhibit greater magnitudes of agonist-induced calcium sensitization (3). Despite these many well-documented differences between fetal and adult patterns of cerebrovascular reactivity, the mechanistic basis for these differences remains unclear.

One important factor that may contribute to age-dependent differences in cerebrovascular reactivity is the corresponding difference in cGMP metabolism. For example, expression of soluble guanylate cyclase, the enzyme responsible for cGMP synthesis, is greater in immature than mature rat pulmonary arteries (5) and also greater in ovine cerebral arteries (29), which probably contributes to the finding that cGMP levels are significantly greater in immature than in mature ovine cerebral arteries (20, 30). The rates of cGMP synthesis in response to maximal concentrations of nitric oxide are also much greater in immature than mature ovine cerebral arteries (30). Rates of hydrolysis of cGMP by phosphodiesterase vary with age as well and generally correlate closely with total soluble guanylate cyclase activity in both rats (8) and sheep (30). Intracellular cGMP concentrations increase more quickly in response to agonist stimulation and attain higher final values in immature than in mature cerebral arteries, suggesting that the influence of cGMP on contractility may diminish during cerebrovascular maturation.

Interestingly, cGMP and its subsequent activation of protein kinase G (PKG) appear capable of altering vascular tone via a broad variety of mechanisms. One of the oldest proposals (9, 17) regarding the mechanism whereby cGMP induces vasodilatation is based on the observation that cGMP reduces cytosolic calcium concentration in multiple preparations. Early studies (26, 32) in many different tissues focused on the ability of PKG to activate calcium pumping into the sarcoplasmic reticulum. More recent studies (16, 32) further suggested that PKG also stimulates the extrusion of calcium. Via more indirect mechanisms, cGMP activated PKG also appears to lower cytosolic calcium by facilitating membrane hyperpolarization through activation of ATP-sensitive potassium channels (1), activation of delayed rectifier K+ channels (32), and inhibition of membrane L-type calcium channels (10, 32). Together, these findings demonstrate that cGMP is a powerful modulator of vascular calcium levels in many different tissues. Thus the higher levels of cGMP typical of immature arteries may help explain why calcium handling is so different from that in adult arteries.

Aside from effects on calcium concentration, cGMP may also attenuate the calcium sensitivity of vascular contractile proteins as suggested by results obtained in both rat mesenteric artery (15) and porcine cardiac fibers (22). The precise mechanisms whereby this effect occurs remain uncertain but may involve phosphorylation of several key contractile proteins, including telokin, a smooth muscle-specific acidic protein that can induce calcium desensitization by enhancing myosin light chain phosphatase activity (31). The phosphorylation state, and thus the activity of, myosin light chain phosphatase has also been reported (23, 24) to be modulated directly by PKG activity. Again, these findings demonstrate that cGMP is a potent modulator of vascular calcium sensitivity. In turn, the higher levels of cGMP typical of immature arteries may help explain why immature cerebral arteries exhibit much different patterns of agonist-induced calcium sensitization than observed in adult arteries.

The present studies were designed to test the general hypothesis that the effects of cGMP on cytosolic calcium concentration and contractile protein calcium sensitivity vary with age. For these studies, we used common carotid and basilar arteries from term fetal and adult sheep because the effects of age on cGMP metabolism are well documented in these preparations. To monitor the effects of cGMP on cytosolic calcium concentrations, we used fura 2-loaded vascular preparations. To observe the effects of cGMP on calcium sensitivity, we used both fura 2-loaded and beta -escin-permeabilized preparations. To minimize complications caused by cGMP metabolism and permeability limitations, we used the nonmetabolizable and highly cell-permeant derivative of cGMP 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP). Together, these approaches enabled a direct assessment of the effects of maturation on the main mechanisms mediating cGMP-induced cerebral vasodilatation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General methods. This study was performed in accordance with all rules and regulations governing the care and use of laboratory animals, as judged by the Institutional Animal Care and Use Committee of Loma Linda University.

Segments of common carotid and basilar arteries were taken from term fetal (138-141 days) and nonpregnant adult sheep 18-24 mo old that were euthanized with an overdose of pentobarbital sodium (60 mg/kg iv). The arteries were cleaned of adhering tissues, cut into ~3- to 4-mm-long segments, and mounted on wires suspended between a force transducer (model TRN011; Kent) and a post attached to a micrometer used to control passive stretch. To avoid possible endothelium-mediated effects, the endothelium was removed by gently rotating each arterial segment around the mounting wires several times to gently scrape the entire luminal surface. The arteries were then immersed in 5-ml jacketed tissue baths, stretched to optimal diameters, and equilibrated for at least 30 min at 38oC (normal ovine core temperature) in a Krebs buffer solution containing (in mM) 122 NaCl, 25.6 NaHCO3, 5.56 dextrose, 5.17 KCl, 2.49 MgSO4, 1.60 CaCl2, 0.114 ascorbic acid, and 0.027 EGTA, and continuously bubbled with 95% O2-5% CO2. To assure inhibition of endothelial release of nitric oxide, all buffer solutions also contained 100 µM NG-nitro-L-arginine methyl ester (L-NAME) and 100 µM NG-nitro-L-arginine (L-NNA). Ablation of endothelial function was verified by testing the vasodilator responses to 1 µM ADP in the presence of 10 µM 8-phenyltheophylline, as previously described (25). During all of the experiments, contractile tensions were continuously digitized, normalized, and recorded with the use of an on-line computer.

Optimal diameters and stretch ratios were determined in fetal and adult segments of ovine basilar and carotid arteries by using methods previously described (11). Briefly, artery segments were initially mounted at near-zero tensions with just enough applied force (<= 600 mg) to remove any vessel slack. Diameters at these near-zero tensions were defined as the D0 diameters. The arteries were then contracted by exposure to an isotonic potassium Krebs solution containing 120 mM K+ and 5 mM Na+. After each exposure to potassium Krebs, the arteries were washed and reequilibrated in normal sodium Krebs. When stable baseline tensions had been attained, the arteries were stretched until basilar and carotid diameters had increased by 5 or 20% of D0, respectively. When baseline tensions had again stabilized, the arteries were contracted once more with potassium Krebs, washed, reequilibrated, and stretched again. This cycle was repeated until the magnitudes of potassium-induced contractions were decreasing with each consecutive stretch. The diameter at which the largest contractile response to potassium Krebs was obtained was defined as optimum diameter, and the ratio of this diameter to D0 was defined as the optimum stretch ratio.

Concentration-relaxation relations for 8-pCPT-cGMP. Because cGMP is readily metabolized within the cell and does not readily cross the cell membrane, we used the highly permeable, nonmetabolizable cGMP analogue 8-pCPT-cGMP (13) for these studies. Initial tone was produced by exposure to either 120 mM potassium or 1 µM serotonin. These concentrations corresponded to a near-maximal concentration (EC90) and half-maximal concentration (EC50), respectively, in both fetal and adult arteries. Once initial contractile tensions were stable, 8-pCPT-cGMP was added in cumulative one-half log concentrations from 1 nM to 1 mM. When maximal relaxation responses had been obtained in all experimental groups, the data were fitted to the logistic equation as previously described (4) to obtain values for pD2 (-log EC50) and maximum contractile tension normalized relative to the maximum response to 120 mM potassium (Emax). An additional group of arteries was pretreated with Rp-8-pCPT-cGMP, an antagonist of PKG (6), before determination of the 8-pCPT-cGMP dose-response relations to verify that the responses observed were PKG dependent.

Effects of 8-pCPT-cGMP on cytosolic calcium concentration in fura 2-loaded arteries. To enable monitoring of cytosolic calcium, basilar artery segments were incubated with the calcium-sensitive fluorescent dye fura 2-acetoxymethyl ester (AM) (5 µM) premixed with pluronic F127 (final concentration 0.01%) for 4 h at 25°C under protection from light. Only basilar artery segments were used in these studies because carotid segments were generally too thick to allow uniform loading and monitoring of fura 2. After loading was completed, the segments were washed and mounted in a fluorometer (model CAF-110, Japan Spectroscopic) that alternately illuminated the segments with two excitation wavelengths of 340 and 380 nm. The fluorescence emitted from the preparation was collected into a photomultiplier circuit through a 500-nm filter. The ratio of the energies emitted at 340 and 380 nm (F340:F380) was calculated automatically as a measure of intracellular calcium and was simultaneously registered along with isometric tension. Given that in previous studies (4) the time course and peak magnitude of contractile response to 120 mM potassium and 1 µM serotonin were not altered after fura loading, any possible acidification of the cells as a result of formaldehyde release from AM hydrolysis (28) appeared to have been negligible. To distinguish the fura 2 calcium signal from autofluorescence or movement artifacts, the energies emitted at F340 and F380 were always monitored separately in addition to measurements of their ratio. Only those preparations in which F340 and F380 changed as mirror images of one another were used. After fura 2 loading, control responses were obtained in all segments. For these responses, the arteries were contracted with serotonin or potassium, then returned to baseline. The segments were then incubated for 40 min with varying concentrations of 8-pCPT-cGMP. Separate time control experiments, in which no 8-pCPT-cGMP was added during the incubation period, were also carried out. After the incubation period, responses to serotonin and potassium were again obtained. After completion of this protocol, the minimum fluorescence was obtained by incubation of the preparations in calcium-free solution containing 140 mM potassium, 2 mM EGTA, and 10 µM ionomycin (at pH 8.6 to optimize the ionomycin effect). After the minimum signal ratio was determined, the vessels were incubated with excess calcium (10 mM) to obtain the maximum signal ratio. All of the fluorescence measurements were then corrected for autofluorescence. The value of the corrected F340:F380 ratio observed during the initial response to potassium or serotonin was defined as 100%, and all subsequent ratio values measured in the same preparation were normalized relative to this value. All procedures have been described in detail in previous publications from our laboratory (4).

Effects of 8-pCPT-cGMP on myofilament calcium sensitivity in fura 2-loaded arteries. To quantify myofilament calcium sensitivity, contractile tensions were measured simultaneously along with cytosolic calcium concentrations in all fura 2 experiments. The resulting contractile tensions were normalized relative to the corresponding maximum initial response, and then myofilament calcium sensitivity was calculated as the percent maximal tension divided by the percentage of the maximal Ca2+ signal ratio. As for calcium concentration, the effects of multiple concentrations of 8-pCPT-cGMP on Ca2+ sensitivity were compared between treatment and control groups for both fetal and adult basilar arteries.

Effects of 8-pCPT-cGMP on myofilament calcium sensitivity in permeabilized arteries. Given that calcium concentration and tension development exhibit much different time courses of response to contractile stimuli, estimates of calcium sensitivity in dynamic non-steady-state preparations can include greater error than that typical of steady-state preparations. For this reason, the estimates of myofilament calcium sensitivity obtained in the dynamic fura 2 preparations were corroborated with steady-state estimates obtained in permeabilized arteries. Arterial segments designated for permeabilization were first equilibrated for 30 min in a relaxing solution that contained (in mM) 5 EGTA, 5 ATP, 110 potassium acetate, 6 magnesium acetate, 1,4-dithiothreitol (DTT), and 20 HEPES, at pH 6.8 (titrated with KOH). The detergent beta -escin was then added at optimum concentrations (basilar: 100 µg/ml, carotid: 150 µg/ml), and incubation was continued for ~30 min at 25°C as previously described (3). Contractile tensions were monitored during skinning, and when tensions attained a magnitude equivalent to that produced by 120 mM potassium before skinning, permeabilization was halted. The skinning solution was then replaced with relaxing solution containing 1 µM A23187, and the arteries were incubated in this solution for 20 min at 25°C to deplete the sarcoplasmic reticulum of calcium. Depletion was verified by complete absence of any contractile response to 10 µM inositol 1,4,5-trisphosphate. After calcium depletion, the vessel preparations were equilibrated for 15 min in a relaxing solution containing 0.1 mM EGTA to minimize diffusion times in high EGTA buffers.

While the effects of standardized concentrations of 8-pCPT-cGMP on myofilament calcium sensitivity were examined in the fura 2 experiments, a different approach was taken in the permeabilized preparations. In these experiments, the magnitude of the relaxation response was normalized instead of the magnitude of the stimulus (8-pCPT-cGMP). This approach offered several advantages; the most important advantage allowed evaluation of the effects of 8-pCPT-cGMP on myofilament calcium sensitivity in a single protocol. The fura 2 experiments required multiple protocols, one for each concentration of 8-pCPT-cGMP employed. In addition, by administering the concentration of 8-pCPT-cGMP (the EC90 concentration) necessary to obtain a near-maximal (90%) relaxation, we were also able to directly compare the extent to which changes in myofilament calcium sensitivity contribute to near-maximal cGMP-induced relaxation in both fetal and adult arteries.

As indicated by a wide variety of studies, myofilament calcium sensitivity is physiologically regulated (3) and is at its minimum basal level in resting, uncontracted arteries. To determine the effects of cGMP on basal myofilament calcium sensitivity, we exposed the arteries to a series of calcium-EGTA buffers that progressively increased calcium concentrations. These buffers were prepared by mixing various ratios of calibrated zero and 10 µM calcium buffers, as previously described (2, 3). The arteries were then incubated for 40 min with varying concentrations of 8-pCPT-cGMP, after which the calcium concentration-response determinations were repeated. Separate time control experiments were also performed, in which no 8-pCPT-cGMP was added between consecutive dose-response determinations. The resulting concentration-response data were then normalized relative to maximum response and fit to the logistic equation to obtain pD2 values for calcium, as previously described (3). These pD2 values were compared between arteries of different age, type, and treatment to determine the effects of these variables on the ability of cGMP to alter basal calcium sensitivity.

In numerous preparations, contractile agonists increase contractile force both by increasing cytosolic calcium concentration and also by increasing the sensitivity of the contractile myofilaments to calcium (3). To assess the ability of cGMP to influence this agonist-induced calcium sensitization, arteries were first briefly exposed to calcium buffer containing 10 µM calcium to obtain a maximum response, after which they were returned to relaxing solution and equilibrated for 40 min with or without varying concentrations of 8-pCPT-cGMP. Following this incubation, 0.3 µM calcium (the EC30 concentration determined in the basal calcium sensitivity concentration-response experiments) was added and once responses had stabilized, graded concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) were added until maximum responses were obtained. GTPgamma S was added to maximally activate all receptor-coupled G proteins and thereby maximally enhance myofilament calcium sensitivity. Under these conditions, where the calcium concentration is held constant, increases in force can be attributed only to increases in myofilament calcium sensitivity. When all of the data had been collected, the responses to GTPgamma S were normalized relative to the maximum initial responses to 10 µM calcium and fit to the logistic equation to obtain pD2 values for GTPgamma S. These pD2 values were compared between arteries of different age, type, and treatment to determine the effects of these variables on the ability of cGMP to alter agonist-stimulated calcium sensitivity.

Data analysis. Distribution analyses were performed on all data sets before any statistical comparisons, and in cases where distributions were not normal, the data were log transformed. After distribution analysis, multifactorial ANOVA analyses were performed with age, artery type, and treatment as factors. Within each ANOVA data set, we ran Bartlett's test to verify homogeneity of variance within each data set (homoscedasticity). When heterogeneous variances were detected, the distributions were normalized via log transformation. This approach produced normally distributed data sets for all analyses. For all comparisons, power analyses were performed routinely to enable reliable conclusions that no significant differences existed for probability values >0.05. All concentration-response relations were analyzed using nonlinear regression with least-squares minimization to obtain values of pD2 (-log EC50) and Emax. The apparent dissociation constant for Rp-8-pCPT-cGMP acting at PKG was determined by Schild analysis. Myofilament calcium sensitivity was calculated as percent maximum force divided by the corresponding percent maximum cytosolic calcium concentration, where maximum force and calcium values were defined under pretreatment control conditions during contractions with either 120 mM potassium or 1 µM serotonin. Values obtained for myofilament calcium sensitivity following treatment with 8-pCPT-cGMP were normalized relative to those observed during pretreatment control contractions. All of the values are given as means ± SE, and n refers to the number of animals. Statistical significance was taken at P < 0.05 level unless otherwise indicated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We obtained 84 basilar segments and 75 common carotid segments from 39 near-term fetuses in these studies. Similarly, 44 young adult sheep provided 90 basilar segments and 82 common carotid segments. Unstressed artery diameters averaged 786 ± 66 and 851 ± 72 µm in fetal and adult basilars, respectively. Corresponding common carotid values averaged 2,351 ± 202 and 3,032 ± 145 µm. Optimal stretch ratios were significantly less in fetal (1.41 ± 0.03, n = 6) than adult (1.55 ± 0.05, n = 5) basilars and were also less in fetal (1.49 ± 0.04, n = 5) than adult (2.01 ± 0.06, n = 5) common carotids. The maximum contractile tensions produced by 120 mM potassium averaged 1.45 ± 0.28 and 2.98 ± 0.43 g in fetal and adult basilars, respectively. Corresponding common carotid values averaged 2.79 ± 0.16 and 14.58 ± 1.35 g.

Concentration-relaxation relations for 8-pCPT-cGMP. When precontracted with potassium, fetal arteries were significantly more sensitive to the relaxant effects of cGMP than were adult arteries, as indicated by the pD2 values (-log EC50) of the concentration-response relations for 8-pCPT-cGMP. These pD2 values were significantly greater in fetal (4.38 ± 0.07) than adult (4.03 ± 0.05) basilars (Fig. 1). After contraction with serotonin, fetal arteries were again significantly more sensitive to cGMP than were adult arteries, and these latter pD2 values averaged 4.92 ± 0.08 and 4.69 ± 0.03, respectively. In addition, sensitivity to cGMP was significantly greater in serotonin-contracted than in potassium-contracted basilar arteries.


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  		Fig. 1.   Concentration-relaxation relations for 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in fetal and adult basilar arteries. Contractile tensions induced by 1 µM 5-hydroxytryptamine (5-HT) (bottom) or 120 mM K+ (top). Inset: bar graphs of pD2 values (-log EC50), significantly greater in fetal (F) than adult (A) arteries in all cases. In addition, pD2 values were significantly greater in 5-HT-contracted than in K+-contracted arteries regardless of age. Maximum percent relaxation (Emax) values did not differ significantly between fetal and adult basilar arteries for either method of contraction. Values are means ± SE for n = 5 in all groups; *significant differences at P < 0.05.

Common carotid results paralleled those obtained in basilar arteries. In potassium-contracted common carotids, fetal arteries were significantly more sensitive to the relaxant effects of cGMP than were adult arteries; pD2 values averaged 4.37 ± 0.03 and 3.98 ± 0.04, respectively. In serotonin-contracted carotids, fetal arteries were again significantly more sensitive to cGMP than were adult arteries, and pD2 values averaged 4.84 ± 0.07 and 4.28 ± 0.07, respectively. Sensitivity to cGMP was also significantly greater in serotonin-contracted than in potassium-contracted common carotid arteries.

In common carotid segments, pretreatment with the PKG inhibitor Rp-8-pCPT-cGMP produced a concentration-related right shift in the 8-pCPT-cGMP concentration-response relation. The apparent dissociation constant for 8-pCPT-cGMP, as determined by Schild analysis of the 8-pCPT-cGMP concentration-response relations, averaged 5.9 ± 0.2, which suggests that a major component of the observed relaxant effect produced by 8-pCPT-cGMP was mediated by PKG (27).

Effects of 8-pCPT-cGMP on cytosolic calcium concentration in fura 2-loaded arteries. In fura 2-loaded basilar arteries, 120 mM potassium-induced increases in cytosolic calcium were not significantly affected by 8-pCPT-cGMP at any concentration examined (25 µM, fetal EC30; n = 6 fetuses, n = 6 adults; 172 µM, fetal EC90; n = 6 fetuses, n = 5 adults; 410 µM, adult EC90; n = 6 adults). This lack of effect was observed in both fetal and adult basilar arteries (Fig. 2A). In contrast, when similar preparations were contracted with 1 µM serotonin, the associated increases in calcium were significantly attenuated by 6 µM 8-pCPT-cGMP (the fetal EC30) in fetal (Delta  = 32.5 ± 7.6%, n = 8) but not adult (Delta  = 8.2 ± 7.8%, n = 5) basilars (Fig. 2B). At higher concentrations of 8-pCPT-cGMP that produced near-maximal relaxation (EC90 concentrations), cytosolic calcium was significantly depressed in both fetal (Delta  = 58.5 ± 4.6%, n = 6) and adult (Delta  = 42.6 ± 4.9%, n = 6) basilars. These data demonstrate that 8-pCPT-cGMP can decrease cytosolic calcium in serotonin-contracted but not potassium-contracted arteries and that serotonin-induced increases in cytosolic calcium are more sensitive to 8-pCPT-cGMP in fetal than adult arteries.


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  		Fig. 2.   Effects of 8-pCPT-cGMP on cytosolic calcium in K+ and 5-HT-contracted arteries. Adult and fetal basilar arteries loaded with fura 2 were contracted with either 120 mM K+ or 1 µM 5-HT, then incubated with or without (control) 8-pCPT-cGMP for 40 min, and were again contracted with either K+ or 5-HT. Free cytosolic calcium levels measured via the F340/F380 ratios were normalized relative to the maximum increases in cytosolic calcium induced by potassium or serotonin, before 8-pCPT-cGMP treatment. A: effects of 25 µM 8-pCPT-cGMP on intracellular calcium concentration ([Ca2+]i) in K+-contracted arteries. This concentration corresponds to the EC30 for 8-pCPT-cGMP in fetal arteries contracted with potassium, as shown in Fig. 1. B: effects of 6 µM 8-pCPT-cGMP on [Ca2+]i in 5-HT-contracted arteries. This latter concentration corresponds to the EC30 for 8-pCPT-cGMP in fetal arteries contracted with serotonin, also as shown in Fig. 1. Values are means ± SE; n = 5-8 animals in each group; *significant differences at P < 0.05.

Effects of 8-pCPT-cGMP on myofilament calcium sensitivity in fura 2-loaded arteries. In fura 2-loaded fetal basilar arteries contracted with 120 mM potassium, pretreatment with 25 µM (fetal EC30) 8-pCPT-cGMP significantly reduced myofilament calcium sensitivity (Delta  = 28.6 ± 5.5%, n = 6), as indicated by the ratio of force to cytosolic calcium in these arteries. In contrast, pretreatment with 25 µM 8-pCPT-cGMP had no significant effect on calcium sensitivity (Delta  = 8.3 ± 15.5%, n = 6) in adult basilar arteries (Fig. 3A). At higher concentrations of 8-pCPT-cGMP (EC90) that produced near-maximal relaxations, myofilament calcium sensitivity was significantly depressed in both fetal (Delta  = 69.1 ± 6.5%, n = 6) and adult (Delta  = 75.0 ± 6.1, n = 6) basilars. In a parallel manner, 6 µM (fetal EC30) 8-pCPT-cGMP significantly reduced myofilament calcium sensitivity after contraction with 1 µM serotonin in fetal (Delta  = 41.5 ± 10.1%, n = 8) but not adult (Delta  = 1.3 ± 24.8, n = 5) basilar arteries. At EC90 concentrations of 8-pCPT-cGMP, myofilament calcium sensitivity in serotonin-contracted adult basilar arteries was also significantly reduced (Delta  = 117.1 ± 20.1%, n = 6). These data demonstrate that 8-pCPT-cGMP can decrease myofilament calcium sensitivity in both potassium- and serotonin-contracted arteries of either age group and that sensitivity to this effect is greater in fetal than adult arteries, regardless of method of contraction.


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  		Fig. 3.   Effect of 8-pCPT-cGMP on myofilament calcium sensitivity in K+ and 5-HT-contracted arteries. Adult and fetal basilar arteries loaded with fura 2 were contracted with 120 mM K+ or 1 µM 5-HT, then incubated with or without (control) 8-pCPT-cGMP for 40 min, and again contracted. Myofilament calcium sensitivity was calculated as the percent ratio of maximum tension divided by percent maximum [Ca2+]i. Values obtained for myofilament calcium sensitivity were normalized relative to those observed during the initial contractions before 8-pCPT-cGMP treatment. A: effects of 25 µM 8-pCPT-cGMP on myofilament calcium sensitivity in K+-contracted arteries. This concentration corresponds to the EC30 for 8-pCPT-cGMP in fetal arteries contracted with potassium, as shown in Fig. 1. B: effects of 6 µM 8-pCPT-cGMP on myofilament calcium sensitivity in 5-HT-contracted arteries. This latter concentration corresponds to the EC30 for 8-pCPT-cGMP in fetal arteries contracted with serotonin, also as shown in Fig. 1. Values are means ± SE; n = 5-8 in each group.*Significant differences at P < 0.05.

Effects of 8-pCPT-cGMP on myofilament calcium sensitivity in permeabilized arteries. As we have previously described (3), the extent of permeabilization with beta -escin is reflected by the ratio of the maximum calcium-induced (10 µM) contraction after permeabilization to the maximum potassium-induced (120 mM) contraction obtained before permeabilization. In the present study, this value averaged 98.9 ± 1.0 and 100.0 ± 1.2% for fetal and adult basilar arteries, respectively. Corresponding values in common carotid segments averaged 99.2 ± 0.8 and 103.8 ± 1.1%, respectively. These values indicate that all of the segments used were optimally permeabilized.

Basal myofilament calcium sensitivity, as indicated by the pD2 values for calcium, was significantly greater in fetal (pD2 = 6.77 ± 0.06, n = 10) than adult (pD2 = 6.64 ± 0.03, n = 10) basilar arteries. In addition, treatment with the EC90 concentrations of 8-pCPT-cGMP for 40 min significantly shifted the basal calcium-force relations in basilar arteries to the right for both the fetus and the adult (Fig. 4). After treatment with the EC90 concentrations of 8-pCPT-cGMP, the pD2 values for basal myofilament calcium sensitivity averaged 6.44 ± 0.07 (n = 10) and 6.38 ± 0.07 (n = 11) in fetal and adult basilars, respectively.


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  		Fig. 4.   Effects of 8-pCPT-cGMP on basal myofilament calcium sensitivity in beta -escin-permeabilized basilar arteries. Concentration-response relations for calcium in both fetal (top) and adult (bottom) basilar arteries permeabilized with beta -escin. In these experiments, the concentrations of 8-pCPT-cGMP were selected as those that produced 90% relaxation in both age groups. As indicated in Fig. 1, these concentrations averaged 172 µM in the fetal arteries and 410 µM in adult arteries. Responses obtained in the presence and absence of 8-pCPT-cGMP (40-min incubation) are indicated by broken and solid lines, respectively. Inset: calcium pD2 (-log EC50) values for both control (solid bars) and treated (hatched bars) segments. Values are means ± SE; n = 10 for fetal basilar arteries and n = 11 for adult basilar arteries. *Significant differences between corresponding fetal and adult artery responses. Despite the markedly lower concentration of 8-pCPT-cGMP used in the fetal arteries, the shift in the calcium-force relation was at least as large as that observed in the adult arteries.

Myofilament calcium sensitivity was significantly more sensitive to GTPgamma S in fetal than in adult basilars, as indicated by the corresponding pD2 values obtained for GTPgamma S (Fig. 5). These values averaged 6.64 ± 0.03 (n = 9) and 6.46 ± 0.06 (n = 9) in fetal and adult basilars, respectively. Treatment with the EC90 concentrations of 8-pCPT-cGMP for 40 min significantly shifted the relations between GTPgamma S and myofilament calcium sensitivity to the right in basilar arteries from both the fetus and the adult. After treatment with the EC90 concentrations of 8-pCPT-cGMP, the pD2 values for GTPgamma S averaged 6.28 ± 0.08 (n = 9) and 6.14 ± 0.06 (n = 9) in fetal and adult basilars, respectively.


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  		Fig. 5.   Effects of 8-pCPT-cGMP on agonist-induced myofilament calcium sensitivity in beta -escin-permeabilized basilar arteries. Many agonists enhance contractile tone by increasing myofilament calcium sensitivity. To quantitate this effect, and the actions of 8-pCPT-cGMP, both fetal (top) and adult (bottom) arteries were permeabilized with beta -escin and then contracted with 0.3 µM calcium (EC30). At this constant calcium concentration, guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) was added cumulatively, and the resultant increases in contractile force were taken as measures of agonist-induced myofilament calcium sensitization. Responses obtained in the presence and absence of 8-pCPT-cGMP (40 min incubation) are indicated by broken and solid lines, respectively. The concentrations of 8-pCPT-cGMP used were selected as those that produced 90% relaxation in both age groups (see Fig. 1) and averaged 61 µM in the fetal arteries, and 231 µM in adult arteries. Inset: pD2 (-log EC50) values for GTPgamma S in both control (solid bars) and treated (hatched bars) segments. Values are means ± SE, n = 9 for all groups. Despite the markedly lower concentration of 8-pCPT-cGMP used in the fetal arteries, the shift in myofilament calcium sensitivity was at least as large as that observed in the adult arteries.

Results similar to those observed in the basilar artery preparations were also observed in the common carotid preparations. The EC90 concentrations of 8-pCPT-cGMP significantly decreased basal calcium sensitivity from 6.65 ± 0.05 to 6.28 ± 0.07 (n = 10) in fetal carotids and from 6.22 ± 0.05 to 5.98 ± 0.04 (n = 10) in adult carotids. Again, myofilament calcium sensitivity was significantly more sensitive to GTPgamma S in fetal (6.56 ± 0.05, n = 10) than in adult (6.26 ± 0.06, n = 8) carotids, as indicated by the corresponding pD2 values obtained for GTPgamma S. Treatment with the EC90 concentrations of 8-pCPT-cGMP significantly decreased the pD2 values obtained for GTPgamma S to 6.20 ± 0.04 and 5.92 ± 0.06 in fetal and adult carotids, respectively. The magnitudes of the shifts in GTPgamma S pD2 values produced by the respective EC90 concentrations of 8-pCPT-cGMP did not vary significantly between artery types in either the fetus or adult.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study offers three new findings. First, contractile force produced by either potassium or 5-hydroxytryptamine (5-HT) is more sensitive to cGMP in fetal than adult basilar arteries. Second, potassium-induced increases in cytosolic calcium are resistant to the effects of cGMP, but those produced by 5-HT are sensitive to attenuation by cGMP, and more so in fetal than adult basilar arteries. Third, myofilament calcium sensitivity under both basal and agonist-enhanced conditions is sensitive to attenuation by cGMP, and again more so in fetal than adult basilar arteries.

Published evidence strongly indicates that cGMP effects vasorelaxation via multiple independent mechanisms (7, 15, 31, 32). Because each of these various mechanisms has a characteristic sensitivity to cGMP, it follows that overall vascular sensitivity to cGMP, which may vary from tissue to tissue, reflects the integration of the specific mechanisms involved and their corresponding sensitivities to cGMP. Consistent with this view, contractile sensitivity to cGMP varied significantly with the method of contraction (Fig. 1). Potassium-induced contractile tone was consistently less sensitive to cGMP than was 5-HT-induced tone, suggesting that method of contraction influences the mechanisms whereby cGMP produces relaxation and that the mechanisms governing cGMP-mediated relaxation are somehow less sensitive to cGMP in depolarized preparations than in 5-HT-contracted arteries. Similarly, contractile tone was more sensitive to cGMP in fetal than adult arteries, regardless of method of contraction, further suggesting that the mechanisms mediating cGMP-induced vasorelaxation varied significantly with maturation. Together, these findings raise the question: which mechanisms mediate cGMP-induced vasorelaxation under each set of experimental conditions?

Owing to the considerable evidence indicating that cGMP can reduce vascular cytosolic calcium (9, 17), we directly measured the effects of cGMP on cytosolic calcium concentration using fura 2-loaded basilar preparations. Potassium-induced increases in cytosolic calcium were resistant to cGMP, even at its EC90 concentration, indicating that cGMP-mediated decreases in cytosolic calcium did not contribute to relaxation of potassium-induced tone in either fetal or adult arteries. Conversely, EC90 concentrations of 8-pCPT-cGMP significantly attenuated 5-HT-induced increases in cytosolic calcium in both fetal and adult basilars (Fig. 2), indicating that potassium depolarization eliminated the ability of cGMP to attenuate cytosolic calcium. Such an effect would be expected if cGMP were acting by stimulating calcium extrusion (16, 32), in which case the effect of cGMP would be counterbalanced by increased calcium influx secondary to potassium depolarization-induced increases in L-type calcium channel conductance. Interestingly, at lower concentrations of 8-pCPT-cGMP (6 µM, the fetal EC30 concentration), 5-HT-induced increases in cytosolic calcium were significantly attenuated only in the fetal arteries, further suggesting that the cGMP-dependent mechanisms mediating calcium extrusion and/or sequestration are more sensitive to cGMP in fetal than adult basilar arteries. This latter observation could clearly help explain why 5-HT-induced contractile tone was more sensitive to cGMP in fetal than adult basilar arteries (Fig. 1). However, differential effects of cGMP on cytosolic calcium concentration cannot explain why potassium-induced tone was more sensitive to cGMP in fetal than adult arteries.

Given that cytosolic calcium concentration and myofilament calcium sensitivity are the two main determinants of contractile force in all smooth muscles (4), it follows that if cGMP produces relaxation independent of effects on cytosolic calcium, then it must in some way influence myofilament calcium sensitivity. In support of this possibility, some reports (15, 22) suggest that cGMP can attenuate myofilament calcium sensitivity. To examine the hypothesis that cGMP influences myofilament calcium sensitivity in ovine basilar arteries, we estimated calcium sensitivity by calculating the ratio of contractile force to cytosolic calcium concentration with the data obtained from each of our fura 2 protocols. EC90 concentrations of cGMP significantly decreased calcium sensitivity in potassium-contracted basilar arteries from both fetal and adult sheep, suggesting that effects on calcium sensitivity were the predominant mechanism mediating relaxation to cGMP in potassium-contracted arteries. Because lower concentrations of cGMP (25 µM, fetal EC30 in potassium-contracted arteries) significantly reduced myofilament calcium sensitivity in fetal but not adult basilar arteries (Fig. 3), the data further suggest that calcium sensitivity is more sensitive to cGMP in fetal than adult arteries. This latter observation could easily explain why potassium-induced tone was more sensitive to cGMP in fetal than adult basilar arteries (Fig. 1).

The analogue 8-pCPT-cGMP attenuated myofilament calcium sensitivity not only in potassium-contracted arteries but also in 5-HT-contracted arteries. At low concentrations (6 µM; fetal EC30 in 5-HT-contracted arteries) 8-pCPT-cGMP significantly decreased myofilament calcium concentration but only in the fetal basilars, again indicating that calcium sensitivity is more sensitive to cGMP in fetal than adult arteries. At EC90 concentrations, 8-pCPT-cGMP also significantly depressed calcium sensitivity in adult arteries, indicating that this effect of cGMP also contributes to relaxation of 5-HT-induced contractile tone in adult arteries. Together, the data suggest that cGMP modulates myofilament calcium sensitivity regardless of age or method of contraction.

While the ratio of contractile force to cytosolic calcium concentration provides a valuable index of myofilament calcium sensitivity in fura 2-loaded preparations, simultaneous transients in both force and calcium that typically occur in such preparations complicate analysis of the relations between these variables. Although the methods of analysis we employed yielded highly consistent estimates of myofilament calcium sensitivity, we decided to corroborate the fura 2 findings by using an independent method. For this approach, we used beta -escin-permeabilized basilar and carotid arteries. A key advantage of this approach was that it enabled determination of steady-state responses in force of contraction to steady-state (buffered) concentrations of calcium. In addition, this approach also enabled differentiation between basal myofilament calcium sensitivity, which is characteristic of resting uncontracted arteries, and agonist-enhanced calcium sensitivity, which is characteristic of myofilaments in arteries contracted with agonists of heterotrimeric G proteins (2, 3). Finally, this approach also enabled evaluation of the effects of cGMP on calcium sensitivity in both basilar and carotid arteries.

Estimates of basal calcium sensitivity came from calcium concentration force curves in beta -escin permeabilized arteries. In general, the effects of 8-pCPT-cGMP on basal calcium sensitivity were consistent with the fura 2 results and indicated that the EC90 concentrations of 8-pCPT-cGMP decreased basal sensitivity to calcium (as indicated by calcium pD2 values) to a similar extent in both fetal and adult basilars (Fig. 4). Because the concentrations of 8-pCPT-cGMP required to obtain this equivalent effect were more than twofold less in fetal (172 µM) than adult (410 µM) basilars, these data demonstrate that basal myofilament calcium sensitivity was more sensitive to cGMP in fetal than adult basilars. Regarding agonist-enhanced myofilament calcium sensitivity, EC90 concentrations of 8-pCPT-cGMP also attenuated GTPgamma S-induced increases in calcium sensitivity to an equivalent extent in fetal and adult basilars (Fig. 5). Again, because the concentrations of 8-pCPT-cGMP required to obtain this equivalent effect were threefold greater in adult (231 µM) than in fetal (61 µM) basilars, these data demonstrate that agonist-enhanced myofilament calcium sensitivity was more sensitive to cGMP in fetal than adult basilars. Together with largely similar results obtained in the common carotid arteries, these data emphasize that both basal and agonist-enhanced myofilament calcium sensitivity are more sensitive to cGMP in fetal than adult ovine basilar and carotid arteries.

Overall, the present data reinforce the view that cGMP effects relaxation through modulation of both cytosolic calcium concentration and contractile protein calcium sensitivity and further indicate that sensitivity to cGMP varies with both mechanism of contraction and postnatal age. Because results in basilar and carotid arteries were generally similar, the data also suggest that the observed effects of maturation on cGMP-induced mechanisms of vasorelaxation are not exclusive to basilar, or even cerebral, arteries. In potassium-contracted arteries, cGMP effects relaxation through inhibition of basal myofilament calcium sensitivity, and thus the pD2 values obtained for 8-pCPT-cGMP-induced relaxation of potassium-induced tone reflect the sensitivity of basal myofilament calcium sensitivity to cGMP. In 5-HT-contracted arteries, cGMP effects relaxation through reductions of both cytosolic calcium concentration and agonist-enhanced myofilament calcium sensitivity. Correspondingly, the pD2 values obtained for 8-pCPT-cGMP-induced relaxation of 5-HT-induced tone reflect the sensitivity of these latter mechanisms to cGMP. Because 5-HT-induced tone was far more sensitive to cGMP than was potassium-induced tone, the data suggest that the mechanisms effecting reductions in cytosolic calcium concentration and agonist-enhanced myofilament calcium sensitivity are much more sensitive to cGMP than are the mechanisms mediating reductions in basal myofilament calcium sensitivity. Regarding the effects of age, the mechanisms that mediate cGMP-induced vasorelaxation appear to be similar in fetal and adult arteries, with the main exception that in fetal arteries these mechanisms are much more sensitive to cGMP than in adult arteries. These basic age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult arteries. The reasons why these mechanisms are more sensitive in fetal arteries, however, remain unclear. In light of the results obtained with the PKG inhibitor Rp-8-pCTP-cGMP, it seems probable that most of the observed cGMP effects were mediated by PKG (6, 12), in which case age-related differences in enzyme abundance, location, or target protein availability could easily be involved. In addition, PKG-independent effects might also be involved, particularly in light of growing evidence that such mechanisms may play an important role in responses to cGMP (16). Many additional experiments will be required to differentiate among these possibilities. Nonetheless, the present data clearly establish that the mechanisms mediating relaxation responses to cGMP are variable and may be of greater physiological significance in fetal than adult cerebral arteries, particularly in light of the higher vascular cGMP concentrations that are characteristic of immature cerebral arteries.


    ACKNOWLEDGEMENTS

The authors thank Dr. William Gerthoffer for comments regarding this manuscript. S. Nauli's work partially fulfilled the requirements for a PhD degree in Pharmacology.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-54120, HL-64867 and HD-31266, and a grant by the Loma Linda University School of Medicine.

Address for reprint requests and other correspondence: W. J. Pearce, Center for Perinatal Biology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: wpearce{at}som.llu.edu).

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

Received 23 June 2000; accepted in final form 21 September 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(3):H1019-H1028
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