Mechanisms of cGMP-induced cerebral vasodilatation: contractile agonist and developmental age make a difference

Mechanisms of cGMP-induced cerebral

vasodilatation: contractile agonist and developmental

age make a difference

William J. Pearce*, Surya M. Nauli

Center for Perinatal Biology, Departments of Physiology and Pharmacology,

Loma Linda University School of Medicine, Loma Linda, CA 92350, USA

Abstract

In light of observations that maturation dramatically influences cerebrovascular metabolism of

cGMP, the present study examines the hypothesis that the ability of cGMP to produce cerebral

vasodilatation changes during maturation. Specifically, these experiments explore age-related

changes in cGMP’s ability to depress cytosolic calcium concentration and attenuate myofilament

calcium sensitivity. The results obtained in a-toxin-permeabilized and Fura-2-loaded ovine basilar

arteries demonstrate that cGMP produces relaxation by attenuating both the cytosolic calcium

concentration and myofilament calcium sensitivity in ovine basilar arteries. More importantly, these

findings reveal that the vasorelaxant potency and efficacy of cGMP are much greater in immature

than in mature cerebral arteries. Together with the elevated levels of the cGMP characteristic of

immature cerebral arteries, these data implicate cGMP as a major regulator of cerebrovascular tone in

the immature cerebral circulation. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: 8-(4-Chlorophenylthio)-3V, 5V-cyclic monophosphate; Cerebrovascular circulation; Guanosine 3V,5V-cyclic monophosphate; Guanylate cyclase; Rp 8-(4-Chlorophenylthio)-3V, 5V-cyclic monophosphorothioate

1. Introduction

The transition from fetal to adult life exacts many changes in cerebrovascular function

[1]. Compared to mature cerebral arteries, fetal cerebral arteries exhibit reduced force

development, greater sensitivity to most contractile agonists, greater myofilament calcium

0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved.

PII: S0531 -5131 (02 )00209 -1

Abbreviations: cGMP, cyclic guanosine monophosphate; PKG, protein kinase G.* Corresponding author. Tel.: +1-909-558-4325; fax: +1-909-558-4029.

E-mail address: [email protected] (W.J. Pearce).

International Congress Series 1235 (2002) 379–393

sensitivity, and altered patterns of calcium mobilization and distribution [1–7]. Cerebro-

vascular maturation also alters patterns of cerebrovascular relaxation, including elevated

basal concentrations of cGMP and greater rates of cGMP synthesis in response to either

endogenous or exogenous nitric oxide (NO) [8,9]. Expression of soluble guanylate

cyclase, the enzyme responsible for cGMP synthesis, is also greater in fetal than in adult

arteries [9–11], as are the rates of cGMP hydrolysis [12,13]. Despite the enhanced ability

of immature cerebral arteries to increase cytosolic cGMP concentrations, the ability of

endothelium-dependent vasodilators to relax cerebral arteries is depressed in fetal

compared to adult cerebral arteries. One possible explanation of this finding is that the

ability of cGMP to mediate vasorelaxation is down regulated in immature arteries. The

present series of experiments was designed to evaluate this hypothesis.

Given that the relaxant effects of cGMP involve both mechanisms which decrease

cytosolic calcium and mechanisms which decrease myofilament calcium sensitivity, we

examined cGMP-induced changes in both cytosolic calcium and myofilament calcium

sensitivity in these experiments. Given that regulation of contractile tone varies consid-

erably for depolarization-induced (receptor-independent) and agonist-induced (receptor-

dependent) contractions, we examined the effects of cGMP on both potassium- and

serotonin-induced contractile tone. In addition, comparing responses in the arteries from

term-fetal lambs and nonpregnant adult sheep assessed the effects of maturation.

2. Effects of 8-pCPT-cGMP on K+ and 5HT-induced tone

Our first approach was to examine the concentration-related effects of a non-metab-

olizable cell-permeant cGMP analogue, 8-pCPT-cGMP, on contractile tone in fetal and

Fig. 1. Concentration–response curves for 8-pCPT-cGMP in basilar arteries contracted with potassium and 5HT.

In arteries precontracted with 5HT, the pD2 ( =� log [EC50]) averages (inset bar graphs) of the concentration–

response relations for 8-pCPT-cGMP were significantly greater in fetal than adult basilars. In arteries precontracted

with potassium, the pD2 averages were also significantly greater in fetal than adult basilars. These findings suggest

that the fetal arteries are more sensitive to cGMP than the corresponding adult arteries. Vertical error bars indicate

standard errors for n= 5 in all groups, and asterisks indicate significant differences via ANOVA at P< 0.05.

W.J. Pearce, S.M. Nauli / International Congress Series 1235 (2002) 379–393380

adult basilar arteries. In these experiments, artery segments were contracted with the age-

specific EC50 and EC95 concentrations for 5HT and potassium, respectively. Once the

initial contractile tensions were stable, 8-pCPT-cGMP was added in cumulative half-log

concentrations from 1 nM to 1 mM [14]. As shown in Fig. 1, 8-pCPT-cGMP significantly

inhibited the contractile tone produced by both potassium and 5HT. In addition, sensitivity

to 8-pCPT-cGMP was significantly greater in fetal compared to adult arteries, as indicated

by the obtained pD2 (� log EC50) values of the concentration–response curves.

To further examine the age-related differences in sensitivity to cGMP, we carried out

concentration–response experiments for 5HT in the control arteries, and in arteries

pretreated with the EC30 concentration of 8-pCPT-cGMP for 5HT contractions (6 AM),

as determined in the experiments shown in Fig. 1. In these experiments, pretreatment with

8-pCPT-cGMP right-shifted the concentration–response relations for 5HT; however, this

shift was significant only in the fetal arteries (Fig. 2). Similarly, pretreatment with the EC30

concentration of 8-pCPT-cGMP for potassium contractions (25 AM) right-shifted the

Fig. 2. Effects of 8-pCPT-cGMP on concentration– response curves for 5HT. Pretreatment with 6 AM 8-pCPT-

cGMP (the fetal EC30 concentration for 5HT contractions) significantly right-shifted the concentration– response

relation for 5HT in fetal but not adult basilar arteries, as indicated by changes in the pD2 values (inset bar graphs).

Vertical error bars indicate standard errors for n= 9 in all groups, and asterisks indicate significant differences via

ANOVA at P < 0.05.

W.J. Pearce, S.M. Nauli / International Congress Series 1235 (2002) 379–393 381

concentration–response relations for potassium, but again, this shift was significant only

in fetal arteries (Fig. 3).

Altogether, the concentration–response experiments demonstrate that the reduced

endothelial vasodilator capacity in immature arteries cannot be explained by attenuated

cGMP efficacy, and thus, must be due to the reduced release of endothelial NO and/or

other relaxant factors. In turn, the enhanced reactivity to cGMP in immature arteries may

be attributable to an increased ability to reduce cytosolic calcium through inhibition of

either release or entry and/or stimulation of either extrusion or sequestration.

3. Effects of 8-pCPT-cGMP on K+ and 5HT-induced increases in cytosolic calcium

To explore the possible age-related differences in the ability of cGMP to influence

cytosolic calcium concentrations, we loaded basilar artery segments with the calcium-

Fig. 3. Effects of 8-pCPT-cGMP on concentration– response curves for potassium. Pretreatment with 25 AM8-pCPT-cGMP (the fetal EC30 concentration for potassium contractions) significantly right-shifted the con-

centration–response relation for potassium in fetal but not adult basilar arteries, as indicated by changes in the pD2

values (inset bar graphs). Vertical error bars indicate standard errors for n= 10 in all groups, and asterisks indicate

significant differences via ANOVA at P < 0.05.


sensitive fluorescent dye Fura-2AM (5 AM), and then washed and mounted the arteries in

a fluorometer (CAF-110, Japan Spectroscopic) as previously described [14,15]. The artery

segments were then pretreated with the fetal EC30 concentration of 8-pCPT-cGMP for 5HT

contractions (6 AM), and exposed to half-log increments in 5HT concentration. In these

experiments, 8-pCPT-cGMP depressed the ability of 5HT to elevate cytosolic calcium in

the fetal arteries, as indicated by a significant reduction in the pD2 values for 5HT (Fig. 4).

In contrast, no such depression was evident in the adult arteries treated with 8-pCPT-

cGMP. In similar experiments using potassium to contract the arteries, however, 8-pCPT-

Fig. 4. Effects of 8-pCPT-cGMP on calcium concentration– response curves for 5HT. Pretreatment with 6 AM8-pCPT-cGMP (the fetal EC30 concentration for 5HT contractions) significantly right-shifted the calcium con-

centration– response relation for 5HT in fetal but not adult basilar arteries, as indicated by changes in pD2 values

(inset bar graphs). Vertical error bars indicate standard errors for eight fetal and five adult arteries. Asterisks

indicate significant differences via ANOVA at P < 0.05.


cGMP had no effect on the ability of potassium to elevate cytosolic calcium in either fetal

or adult arteries (Fig. 5).

In light of the marked differences in the ability of 8-pCPT-cGMP to influence 5HT- and

potassium-induced increases in cytosolic calcium, we examined the near-maximal effects

of 8-pCPT-cGMP on the responses of cytosolic calcium to both potassium and 5HT. In

these experiments, the arteries were contracted with the EC50 or EC95 concentrations of

5HT or potassium, then returned to the baseline and incubated for 40 min with the age- and

agonist-specific EC90 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 5HT and potassium were again obtained. As

previously observed, even the EC90 concentrations of 8-pCPT-cGMP had no significant

effects on the responses of cytosolic calcium concentration to potassium in either fetal or

Fig. 5. Effects of 8-pCPT-cGMP on calcium concentration– response curves for potassium. Pretreatment with 25

AM 8-pCPT-cGMP (the fetal EC30 concentration for potassium contractions) had no significant effect on the

calcium concentration– response relation for potassium in either fetal or adult basilar arteries. Vertical error bars

indicate standard errors for eight fetal and five adult arteries.


adult arteries (Fig. 6). In contrast, the EC90 concentrations of 8-pCPT-cGMP significantly

depressed the ability of 5HT to increase cytosolic calcium in both fetal and adult arteries.

4. Effects of 8-pCPT-cGMP on K+ and 5HT-induced increases in myofilament

calcium sensitivity

Given that 8-pCPT-cGMP relaxed the potassium-induced tone (Fig. 1) without altering

the ability of potassium to increase cytosolic calcium (Fig. 5), it follows that 8-pCPT-

cGMP must relax the potassium-induced tone by attenuating myofilament calcium

sensitivity. In addition, because 8-pCPT-cGMP inhibited potassium-induced tone more

in fetal than adult arteries (Fig. 1), it also follows that the effect of 8-pCPT-cGMP on

calcium sensitivity must be greater in fetal than in adult basilar arteries. In order to explore

these possibilities, we again used Fura-2-loaded basilar arteries and simultaneously

measured the cytosolic calcium concentration and contractile force in the presence and

absence of 8-pCPT-cGMP. The myofilament calcium sensitivity was estimated as the ratio

of the change in tension divided by the corresponding change in cytosolic calcium

concentration, as previously described [14]. In one group of such arteries, we monitored

tension and calcium concentrations during exposure to graded concentrations of 5HT in

Fig. 6. Effects of EC90 concentrations of 8-pCPT-cGMP on K- and 5HT-induced increases in cytosolic Ca2 +

concentration. Pretreatment with age- and agonist-specific EC90 concentrations of 8-pCPT-cGMP had no

significant effect on the ability of the EC95 concentration of potassium to increase cytosolic calcium

concentration. In contrast, the EC90 concentrations of 8-pCPT-cGMP significantly attenuated the ability of the

EC50 concentration of 5HT to increase cytosolic calcium concentration. Vertical error bars indicate standard errors

and asterisks indicate significant differences via ANOVA at P < 0.05.


the presence and absence of 6 AM 8-pCPT-cGMP (the fetal EC30 for 5HT contractions).

All of the arteries were incubated with either 8-pCPT-cGMP or vehicle for 30 min prior to

exposure of the first 5HT concentration. In the fetal arteries, 8-pCPT-cGMP attenuated the

ability of 5HT to increase myofilament calcium sensitivity, as indicated by a significant

decrease in the pD2 values for 5HT in the treated arteries (Fig. 7). Treatment with 8-pCPT-

cGMP also significantly decreased the maximum efficacy of 5HT toward calcium

sensitivity in the fetal arteries. In contrast, 8-pCPT-cGMP had no significant effect on

the calcium sensitivity responses to 5HT in the adult basilar arteries.

In an additional group of arteries, we monitored tension and calcium concentrations

during exposure to graded concentrations of potassium in the presence and absence of 25

AM 8-pCPT-cGMP (the fetal EC30 for potassium contractions). Again, 8-pCPT-cGMP

Fig. 7. 8-pCPT-cGMP can attenuate 5HT-induced increases in Ca-sensitivity. Basilar arteries were loaded with

Fura-2AM, then incubated 30 min with either 6 AM 8-pCPT-cGMP (the fetal EC30 concentration for 5HT

contractions) or vehicle, and then exposed to graded concentrations of 5HT. The resulting changes in tension were

divided by corresponding increases in cytosolic calcium concentration to estimate changes in myofilament

calcium sensitivity. The ability of 5HT to enhance calcium sensitivity was depressed by 8-pCPT-cGMP as

reflected by change in both the potency (inset bar graphs) and efficacy of 5HT. Vertical error bars indicate

standard errors and asterisks indicate significant differences via ANOVA at P < 0.05.


depressed both the potency and efficacy of the potassium-induced changes in myofilament

calcium sensitivity in the fetal arteries, but was without significant effect in the adult

arteries (Fig. 8), at least at the fetal EC30 concentration of 8-pCPT-cGMP.

To confirm the ability of 8-pCPT-cGMP to influence myofilament calcium sensitivity,

we examined calcium concentration–response relations in permeabilized basilar arteries.

The arteries were permeabilized with a-toxin at 10 Ag/ml as previously described [3,14],

and exposed to graded concentrations of buffered calcium in the presence and absence of

Fig. 8. 8-pCPT-cGMP can attenuate potassium-induced increases in Ca-sensitivity. The effects of 8-pCPT-cGMP

on potassium-induced changes in myofilament calcium sensitivity were determined as described in Fig. 7, with

the exception that 25 AM 8-pCPT-cGMP (the fetal EC30 concentration for potassium contractions) was used, and

concentration–response relations were determined for potassium. The ability of potassium to enhance calcium

sensitivity was depressed by 8-pCPT-cGMP as reflected by change in both the potency (inset bar graphs) and

efficacy of 5HT. Vertical error bars indicate standard errors and asterisks indicate significant differences via

ANOVA at P < 0.05.


25 AM 8-pCPT-cGMP (the fetal EC30 for potassium contractions). Under these con-

ditions, 8-pCPT-cGMP significantly reduced the myofilament calcium sensitivity in the

fetal arteries, as indicated by a significant reduction in the pD2 concentration for calcium

(Fig. 9). Treatment with 8-pCPT-cGMP, however, had no significant effect on the

calcium–force relation in the adult arteries. These data demonstrate that basal myofila-

ment calcium sensitivity is more potently attenuated by 8-pCPT-cGMP in fetal than in

adult basilar arteries.

Because myofilament calcium sensitivity is often dramatically enhanced by hetero-

trimeric G-protein receptor activation [3,16], it is possible that cGMP-mediated influences

on calcium sensitivity may differ under basal and agonist-stimulated conditions. In order

to explore this idea, we contracted permeabilized arteries with their EC30 concentration of

calcium, and then added the non-metabolizable G-protein activator, GTPgS, to baths in

Fig. 9. 8-pCPT-cGMP shifts the basal Ca– force concentration–response relation. Pretreatment with 25 AM8-pCPT-cGMP (the fetal EC30 concentration for potassium contractions) significantly right-shifted the basal

calcium–force concentration–response relations obtained in permeabilized fetal basilar arteries, as indicated by a

significant reduction in the pD2 concentration of calcium (inset). In contrast, pretreatment with 25 AM 8-pCPT-

cGMP had no significant effect on basal calcium–force concentration–response relations in adult basilar arteries.

Vertical error bars indicate standard errors and asterisks indicate significant differences via ANOVA at P < 0.05.


stepwise cumulative increments. As previously reported [16], the addition of GTPgS

produced a concentration-related increase in myofilament calcium sensitivity. Pre-equili-

bration with the age-dependent EC90 concentrations of 8-pCPT-cGMP (fetal EC90 = 172

AM; adult EC90 = 410 AM) significantly reduced sensitivity to GTPgS in both age groups,

indicating that cGMP influences not only basal, but also agonist-enhanced myofilament

Fig. 10. 8-pCPT-cGMP attenuates the ability of GTPgS to enhance Ca-sensitivity. Permeabilized basilar arteries

were precontracted with their EC30 concentration of calcium, and then exposed to graded cumulative

concentrations of GTPgS, a non-metabolizable activator of G-proteins. GTPgS increased the myofilament

calcium sensitivity in a concentration-dependent manner, and these increases were attenuated by pretreatment

with the EC90 concentrations of 8-pCPT-cGMP in both fetal (172 AM) and adult (410 AM) arteries. Vertical error

bars indicate standard errors and asterisks indicate significant differences via ANOVA at P < 0.05.


Fig. 11. Effects of maturation on mechanisms of cGMP-dependent vasodilatation. In the above diagram, a circled

M with a plus sign indicates enhancement by maturation, whereas a circled M with a minus sign indicates

reduced activity or expression during maturation. Compared to the adult arteries, immature cerebral arteries

typically exhibit a reduced eNOS abundance and capacity for endothelial NO release [17], which is partially

offset by an increased abundance and activity of soluble guanylate cyclase [18]. Levels of cGMP are elevated in

fetal compared to adult cerebral arteries, and this can produce changes in cAMP levels through actions on

cGMP-sensitive phosphodiesterases [19]. The predominant effect of cGMP on vascular tone, however, is

mediated through activation of protein kinase G, which exists in at least two isoforms [20]. The effect of

maturation on the abundance and activity of PKG isoforms in cerebral arteries is, at present, unknown. PKG can

potentially lower calcium concentration through inhibition of release through phosphorylation of IRAG, a

specialized protein in the sarcoplasmic reticulum [25]. By increasing the activity of calcium-sensitive potassium

channels, PKG can also hyperpolarize the sarcolemma and further limit calcium entry [26]. PKG may also

stimulate extrusion and sequestration directly [27,28]. Regarding the calcium sensitivity, PKG might

phosphorylate proteins such as telokin [29,30], myosin light chain phosphatase [30,31], Rho kinase [32],

SM22-a [33], and possibly even HSP-20 [34].


calcium sensitivity (Fig. 10). Because similar reductions in sensitivity to GTPgS were

achieved with much lower concentrations of 8-pCPT-cGMP in fetal (172 AM) than in the

adult arteries (410 AM), these data also demonstrate that agonist-enhanced calcium

sensitivity is more sensitive to cGMP in fetal than in adult arteries.

5. Overview

Whereas it is well established that immature cerebral arteries exhibit a depressed

capacity for endothelial NO production [17] that is offset in part by an increased

abundance of soluble guanylate cyclase [18], the present results further suggest that

increased reactivity to cGMP is also characteristic of the immature cerebral arteries.

Certainly, increased basal cGMP concentrations [8,9,11] can have important effects by

modulating cGMP-sensitive phosphodiesterases [19], but the predominant vasoactive

effects of cGMP are probably mediated by protein kinase G [20]. Correspondingly, it is

reasonable to postulate that the greater sensitivity to cGMP typical of the immature arteries

is simply a consequence of the greater abundance of PKG in the fetal compared to the

adult cerebral arteries, as it is in fetal compared to adult hearts [21]. Similarly, the

predominant isoform of PKG [20] may also vary with developmental age.

Downstream from PKG, numerous different proteins could mediate PKG’s effects on

calcium concentration and myofilament calcium sensitivity (Fig. 11). PKG can phosphor-

ylate membrane channels and ion pumps to inhibit calcium influx, inhibit calcium release

from the sarcoplasmic reticulum (SR), and thereby decrease cytosolic calcium concen-

trations [22–26]. PKG can also increase calcium efflux and calcium uptake into the SR

[27,28]. As regards to the possible inhibitory effects on the myofilament calcium

sensitivity, PKG might phosphorylate proteins such as telokin [29,30], myosin light chain

phosphatase [30,31], Rho kinase [32], SM22-a [33], and possibly even HSP-20 [34].

Without doubt, there are many possibilities to explore, particularly because developmental

differences in the relative abundance and activity of almost all of these proteins are largely

unknown. Nonetheless, the present data clearly indicate that cerebrovascular sensitivity to

cGMP is elevated in immature cerebral arteries, a factor that undoubtedly contributes to

the lower resting cerebrovascular resistance characteristic of neonates, particularly when

combined with the elevated levels of cGMP also typical in this age group.

Acknowledgements

USPHS Grants HL54120, HL64867 and HD31266, and the Loma Linda University

School of Medicine supported the work reported here. The authors extend their sincere

appreciation to James Williams for his excellent technical support of this project.

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Mechanisms of cGMP-induced cerebral vasodilatation: contractile agonist and developmental age make a difference