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Am J Physiol Regul Integr Comp Physiol 282: R131-R138, 2002; doi:10.1152/ajpregu.00318.2001
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Vol. 282, Issue 1, R131-R138, January 2002

L-type Ca2+ channels in fetal and adult ovine cerebral arteries

Arlin B. Blood, Yu Zhao, Wen Long, Lubo Zhang, and Lawrence D. Longo

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, we reported that, whereas in cerebral arteries of the adult a majority of norepinephrine (NE)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) comes from release of the sarcoplasmic reticulum (SR) Ca2+ stores, in the fetus the SR Ca2+ stores are relatively small, and NE-induced increase in [Ca2+]i results mainly from activation of plasma membrane L-type Ca2+ channels (20). In an effort to establish further the role of L-type Ca2+ channels in the developing cerebral arteries, we tested the hypothesis that, in the fetus, increased reliance on plasmalemmal L-type Ca2+ channels is mediated, in part, by increased L-type Ca2+ channel density. We used 3H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) to measure L-type Ca2+ channel density (Bmax) in the cerebral arteries, common carotid artery (CCA), and descending aortae of fetal (~140 gestation days), newborn (7-10 days), and adult sheep. In the cerebral and common carotid arteries, Bmax values (fmol/mg protein) of fetuses and newborns were significantly greater than those of adults. Western immunoblotting assay also revealed that the density of L-type Ca2+ channel protein in the cerebral arteries and CCA was about twofold greater in the fetus than the adult. Finally, compared with the adult, fetal cerebral arteries demonstrated a significantly greater maximum tension and [Ca2+]i in response to stimulation with the L-type Ca2+ channel agonist Bay K 8644. In addition, Bay K 8644-stimulated fetal vessels demonstrated a maximal tension and [Ca2+]i similar to that observed in response to stimulation with 10-4 NE. These results support the idea that fetal cerebrovascular smooth muscle relies more on extracellular Ca2+ and L-type Ca2+ channels for contraction than does the adult and that this increased reliance is mediated, in part, by greater L-type Ca2+ channel density. This may have important implications in the regulation of cerebral blood flow in the developing organism.

cerebral circulation; norepinephrine; vascular smooth muscle; intracellular calcium; PN200-110; Bay K 8644; fetus; newborn


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INCREASE IN INTRACELLULAR Ca2+ concentration ([Ca2+]i) required for smooth muscle contraction is a result of influx of Ca2+ through L-type Ca2+ channels as well as the Ins(1,4,5)P3-mediated release of Ca2+ from the sarcoplasmic reticulum (SR). A growing body of evidence suggests that the source of cytosolic Ca2+ during vascular smooth muscle (VSM) contraction changes with development. For instance, we demonstrated that contracting fetal cerebral arteries rely almost entirely on extracellular Ca2+ influx through L-type Ca2+ channels (20). In addition, the SR of fetal VSM cells is poorly developed (4, 29) and contributes minimally to the increase in [Ca2+]i during contraction (19). Adult smooth muscle cells, on the other hand, contain a well-developed SR that, on Ins(1,4,5)P3 receptor activation, contributes a majority of the cytosolic Ca2+ necessary for contraction (31). In addition, adult VSM cells are less dependent on the activation of L-type Ca2+ channels for contraction (20).

Possible mechanisms for increased fetal VSM reliance on extracellular Ca2+ include increased L-type Ca2+ channel density, altered L-type Ca2+ channel voltage sensitivity, increased extracellular [Ca2+], and so forth. In the present study, we tested the hypothesis that in cerebral arteries, smooth muscle L-type Ca2+ channel density decreases as a function of developmental age. We also tested the hypothesis that in the fetus, as a means of supporting its increased reliance on extracellular Ca2+ for VSM contraction, plasma [Ca2+] is relatively high, compared with adult values. Finally, we tested the hypothesis that cerebral arteries in the fetus are more sensitive than those of the adult to stimulation by the L-type Ca2+ channel agonist Bay K 8644.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation for immunoblotting and binding assays. We obtained arterial samples from near term (~140 days gestation) fetal sheep, newborn lambs (7-10 days), and young female nonpregnant and pregnant adults (<2 yr). Sheep were obtained (Nebeker Ranch, Lancaster, CA) and were killed using 100 mg/kg intravenous pentobarbital sodium. Immediately after the sheep were killed, anterior, middle, and posterior cerebral arteries, common carotid artery (CCA), and descending aortae were dissected out and immediately wrapped in aluminum foil, snap-frozen in liquid nitrogen, and stored at -80°C until use. To ensure tissue consistency, descending aortae were always cut from within 5 cm superior to the branch of the renal artery. All surgical and experimental procedures were performed within the regulations of the Animal Welfare Act, and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals was strictly adhered to, as was "The Guiding Principles in the Care and Use of Animals" approved by the Council of the American Physiological Society, and governed by the Animal Care and Use Committee of Loma Linda University.

Radioligand binding assay. CCAs or aortae from one or two adult, two or four newborn, and two or four fetal sheep were pooled separately to obtain ~1.5 g of tissue for each assay. Cerebral arteries from five or six adult, four or five newborn, and four or five fetal sheep were pooled separately to obtain ~0.5 g of tissue for each assay. Tissue samples were ground to a fine powder in liquid N2. The samples were then suspended in 10 ml of binding buffer (50 mM Tris, 1.5 mM MgCl2, 2.4 mM CaCl2, 1 mM phenylmethylsulfonylchloride, 5 mM benzamadine, 1 µM pepstatin A, pH 7.4) using a Polytron tissue homogenizer (Brinkman Instruments, Westbury, NY). The resulting homogenate was centrifuged for 10 min at 1,500 g at 4°C, and the pellet containing unlysed cells and debris was discarded. The supernatant was then centrifuged at 110,000 g (50,000 rpm) for 45 min at 4°C in an ultracentrifuge (model L3-50, Beckman Instruments, equipped with a TI-50 rotor). The resulting pellet containing the cellular membrane fraction was then suspended in 1 ml of binding buffer, and the supernatant was discarded. The protein concentration of each resuspended membrane fraction was determined using the Bradford method (2). Unless otherwise noted, all chemical compounds were purchased from Sigma (St. Louis, MO).

Receptor binding assays were carried out with 60 µg of protein in 1 ml of binding buffer, with the selective L-type Ca2+ channel antagonist 3H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) in concentrations varying from 0.1 to 4.0 nM. Nonspecific binding was measured by incubating the above mixture in the presence of 2 µM of the unlabeled L-type Ca2+ channel antagonist nifedipine. The binding mixture was allowed to incubate in glass tubes for 90 min in the dark at room temperature. At the end of the incubation, the membrane portions were collected on GF/C grade filters (Whatman, Maidstone, UK) in a cell harvester (Brandel, Gaithersburg, MD). The filters were prewetted with rinse buffer (50 mM Tris, 0.01% Triton X-100, pH 7.4) and then washed twice with rinse buffer after sample harvesting. The filters were counted in 5 ml of scintillation cocktail (Scintiverse, Fischer Scientific, Fairlawn, NJ) in a scintillation counter (model 1900CA, Packard Instruments, Downers Grove, IL). Specific binding curves were calculated by subtraction of the nonspecific PN200-110 binding curves from the total PN200-110 binding curve and were then analyzed by use of a nonlinear least-squares regression to fit binding data to a rectangular hyperbola. This fitting generated both the maximal radioligand binding or channel density (Bmax) and the dissociation constant (KD) values (Prism, Graphpad Software, San Diego, CA).

Immunoblotting of L-type Ca2+ channel protein. Fetal and adult sheep cerebral arteries, CCA, and aorta were isolated as described above. Frozen samples were homogenized in liquid N2 with a porcelain mortar and pestle. Homogenized samples were then incubated for 10 min in the lysing buffer (20 mM Tris · HCl, 1 mM EDTA, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.1 mg/ml benzamidine, and 8 µg/ml calpain inhibitors I and II, pH 7.4). Nuclei and debris were pelleted by centrifugation at 100 g for 20 min. The whole cell lysate was then centrifuged for 10 min at 10,000 g. The resulting pellet (membrane-bound protein) was resuspended in the lysing buffer, sonicated, and stored at -20°C.

Western immunoblots were run on 10% polyacrylamide gels. After transfer of the proteins to a nitrocellulose membrane (Fisher Scientific, Tustin, CA) in 10% methanol for 1 h at a constant 100 V, the L-type Ca2+ channel protein was identified using polyclonal rabbit antibodies for the alpha 1c-subunit of the L-type Ca2+ channel (Alamode Labs, Jerusalem, Israel). A goat anti-rabbit antibody with alkaline phosphatase was used to visualize the antibodies. The results were then quantified using a densitometer (Alpha Inotech Imaging System, Rockville, MD).

Plasma free and total Ca2+ levels. Blood samples for measurement of plasma ionized and total Ca2+ levels were obtained from chronically catheterized ewes and fetuses surgically prepared, as previously described (18). Briefly, eight pregnant ewes were surgically instrumented with femoral venous catheters at 122 to 126 days gestation. At the same time, the fetus was exposed through a midline incision in the abdomen of the ewe and instrumented with a polyvinyl catheter in the CCA. The catheter was exteriorized through an incision in the maternal flank and stored in a pouch sutured to the maternal skin. The fetus was then returned to the uterus, and the uterus and abdomen of the ewe were sutured in layers. Five to seven days after surgery, 3-ml blood samples were collected simultaneously from the maternal and fetal catheters into sodium-heparin Vacutainers (Fisher Scientific). The samples were immediately placed on ice and processed by the Loma Linda University Medical Center clinical laboratory for both ionized and total plasma Ca2+ (Synchron CX system, Beckman, Westbury, NY). After collection of the plasma samples, the animals were used for further study in an unrelated protocol.

Effect of Bay K 8644. We removed the fetal or adult brain as described above, placed it in iced saline, and dissected out and cleaned the cerebral arteries. We have shown that this method of death has no significant effect on vessel reactivity compared with use of other anesthetic agents (23). To avoid the complication of endothelial-mediated effects, we removed the endothelium by carefully inserting a small wire three times (21). To confirm endothelium removal, we contracted the vessel with 10-5 M 5-hydroxytryptamine and at the plateau added 10-6 M adenosine diphosphate. Vessels that relaxed >20% after this treatment were rejected for further study. Cerebral arteries were used immediately for simultaneous measurements of the [Ca2+]i and tensions (20).

We cut the middle cerebral arteries (MCAs) into rings 2 mm in length and mounted them on two tungsten wires (0.13-mm diam; A-M Systems, Carlsborg, WA). We attached one wire to an isometric force transducer (Kent Scientific, Litchfield, CT) and the other to a post attached to a micrometer used to vary resting tension in a 5-ml tissue bath mounted on Jasco CAF-110 intracellular Ca2+ analyzer (Jasco, Easton, MD). We measured vascular tension, as previously described (20). MCA rings were equilibrated under 0.3 g tension at 25°C for 40 min before loading with the acetoxymethyl ester of fura 2 (fura 2-AM; Molecular Probes, Eugene, OR), a fluorescent Ca2+ indicator that is a measure of mean cytoplasmic [Ca2+]i (10). Vessels were illuminated alternatively (125 Hz) at excitation wavelengths of 340 and 380 nm by means of two monochromators in the light path of a 75-W xenon lamp. Tissue fluorescence emission was measured at 510 nm by a photomultiplier. Fura 2 fluorescence and force were measured simultaneously at 38°C. During all contractility experiments, we continuously digitized and recorded contractile tensions and the fluorescence ratio (F340/380) using an online computer, as previously described (20).

We first contracted the arterial segment by adding 120 mM isotonic KCl to the Krebs buffer. After peak tension was reached, we washed the artery with normal sodium Krebs solution and allowed it to return to baseline tension for 15 min. Subsequent dose-response curves were obtained with NE or Bay K 8644 at doses increasing in half-log increments (10-9 to 10-4 M and 10-9 to 10 -6 M, respectively). We evaluated the contractile response for tension and fluorescence ratio by measuring the maximum response (a measure of "efficacy") and the pD2 (the negative logarithm of the EC50 for NE or Bay K 8644 and an index of tissue "sensitivity" or "potency") (20).

Statistics. For L-type Ca2+ channel radioligand binding studies, we used vessels from 22 fetuses, 20 newborns, and 14 adult sheep. Tissues from several animals were pooled for each assay as indicated above; n refers to the number of receptor assays performed. Radioligand binding assays were carried out in duplicate for all CCA and aorta samples. Due to limited sample availability, cerebral artery assays were not carried out in duplicate. Differences between pregnant and nonpregnant adults were not significant, so all adult data were pooled. For the immunoblotting assay, vessels from four fetuses and four adults were used. For the plasma [Ca2+] studies, eight adults and their respective fetuses were studied. For vessel tension studies, vessels from 14 different fetal and adult brains were studied with NE stimulation, and vessels from five different brains were studied with Bay K 8644 stimulation. All values were calculated as the means ± SE. Comparisons were made among fetus, newborn, and adult for each vessel type. Significant Bmax differences between age groups were assessed for each vessel type by one-way ANOVA with Newman-Keuls post hoc test. Differences between fetal and adult Western immunoblotting densitometry and plasma [Ca2+] were assessed using Student's t-test. Unless otherwise indicated, statistical significance implies P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

L-type Ca2+ channel density. To determine L-type Ca2+ channel density (Bmax) and affinity (KD) in the three vessel types studied, we performed radioligand binding studies. Figure 1A shows the radioligand binding curves of PN200-110 for fetal, newborn, and adult cerebral arteries. Figure 1B shows the Bmax values for the three vessel types in the three age groups studied. The Bmax values (fmol/mg protein) for fetal, newborn, and adult cerebral arteries (n = 4 each) were 140 ± 12, 124 ± 12, and 58 ± 8, respectively. For fetal, newborn, and adult CCA (n = 6 each), the values were 147 ± 12, 131 ± 11, and 58 ± 5, respectively. The receptor density values for both fetal and newborn cerebral arteries and CCA were significantly greater (~130%) than those of the adult vessels (P <=  0.001). The Bmax values for fetal, newborn, and adult aortae were 104 ± 8, 108 ± 6, and 95 ± 7, respectively, values that were not statistically different. We also observed significantly different receptor densities between vessel types within the same age group. L-type Ca2+ channel density was significantly greater in fetal CCA than in fetal aortae and significantly less in adult cerebral arteries and CCAs than in aortae.


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  		Fig. 1.   A: L-type Ca2+ channel specific binding of 3H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) to fetal, newborn, and adult cerebral arteries. Varying concentrations of PN200-110 were incubated with aliquots of membrane fractions of the cerebral arteries (n = 4); see METHODS for details (*, fetal; , newborn; , adult). B: receptor density (Bmax) values of fetal (cross- hatched bars), newborn (open bars), and adult (solid bars) cerebral arteries, common carotid artery, and aortae. *Significantly greater than adult (P < 0.01). +Significantly different than aorta for that age group (P < 0.05).

The KD values (nM) of PN200-110 for fetal, newborn, and adult cerebral arteries were 0.58 ± 0.15, 0.64 ± 0.18, and 0.38 ± 0.15, respectively. KD values for fetal, newborn, and adult CCA were 0.65 ± 0.14, 0.38 ± 0.21, and 0.33 ± 0.20, respectively. KD values for fetal, newborn, and adult aortae were 0.33 ± 0.19, 0.40 ± 0.17, and 0.36 ± 0.20, respectively. No significant developmental or vessel type differences were observed in the KD values, suggesting the presence of a single type of L-type Ca2+ channel.

Immunoblotting of L-type Ca2+ channels. To determine the relative abundance of L-type Ca2+ channel protein in fetal and adult cerebral arteries, we performed Western immunoblotting analysis using monoclonal antibody against the alpha 1c-subunit of the L-type Ca2+ channel. Figure 2A shows representative immunoblotting results from one experiment for the alpha 1c-subunit of L-type Ca2+ channels in membrane protein fractions from the cerebral arteries and CCAs of fetal and adult sheep. Densitometric measurements normalized using alpha -tubulin levels are presented in Fig. 2B. The data indicate a two- to threefold greater abundance of L-type Ca2+ channel protein for both CCAs and cerebral arteries in the fetus compared with the adult. Similar results were obtained in a total of three experiments. Results for the aorta (n = 2) were also similar to those of the radioligand binding study (data not shown).


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  		Fig. 2.   Western immunoblot (A) and densitometric (B) analysis for the L-type Ca2+ channel alpha 1c-subunit protein in membrane fractions of homogenized fetal (F) and adult (A) common carotid arteries and fetal and adult cerebral arteries. Densitometry measurements were normalized to the density of the alpha -tubulin band for each respective lane (n = 3, see METHODS for details). *Significantly greater than adult.

Plasma [Ca2+] in fetal and adult sheep. To determine the relative concentrations of free ionizable and total Ca2+ in fetal and adult ovine plasma, we quantified these values. Figure 3 depicts the total and ionized [Ca2+] in plasma samples from chronically catheterized fetal and adult sheep (n = 5 each). Fetal values were significantly higher for both ionized (1.4 ± 0.1 vs. 1.2 ± 0.1 mM) and total (2.9 ± 0.2 vs. 2.4 ± 0.1 mM) [Ca2+] (P < 0.01 for each).


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  		Fig. 3.   Plasma ionized and total Ca2+ concentrations (mmol/l) from samples taken from chronically catheterized fetal (crosshatched bars, n = 8) and adult (solid bars, n = 8) sheep. Ewes and fetuses were catheterized and allowed to recover for 5 days before sample collection. *Significantly greater than adult values (P <=  0.01).

Contractile and [Ca2+]i responses to NE and L-type Ca2+ channel activation. To determine the relative responses of fetal and adult cerebral arteries to stimulation by the alpha -adrenergic agonist NE vs. those responses to the L-type Ca2+ channel opener Bay K 8644, we measured the tension and [Ca2+]i in response to these compounds. Figure 4, A and B, shows the contractile tensions and fura 2 fluorescence ratio (F340/380) responses of fetal and adult cerebral arteries in response to NE (10-9-10-4 M). As seen in Fig. 4A, the maximum NE-induced tensions in adult and fetal MCA were 1.6 ± 0.1 and 1.2 ± 0.1 g, respectively. The corresponding pD2 values were 6.1 ± 0.1 and 6.2 ± 0.2, respectively (values shown are means ± SE). As seen in Fig. 4B, there was no significant difference between adult and fetal cerebral arteries in NE-stimulated [Ca2+]i. Maximum responses were 0.15 ± 0.01 vs. 0.16 ± 0.01, respectively. The corresponding pD2 values were 6.6 ± 0.1 for each age group.


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  		Fig. 4.   Norepinephrine (NE) and Bay K 8644 dose-response relationships for adult and fetal main branch middle cerebral arteries (MCA). Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, we induced subsequent contractions using cumulative doses of NE or Bay K 8644 added in half-log increments (see METHODS for details). A: vascular tensions (g) for adult (, solid line) and fetal (black-triangle, dashed line) MCA. Adult and fetal maximum NE-induced tensions were 1.6 ± 0.1 and 1.2 ± 0.1 g, respectively, whereas the negative logarithms of the EC50 values were 6.1 ± 0.1 and 6.2 ± 0.1, respectively. Points shown are means ± SE. B: fluorescence ratios (F340/380) for adult and fetal MCA (symbols same as above). Maximum NE-induced ratios were 0.15 ± 0.02 and 0.16 ± 0.01, respectively. Inset: ratio of NE-induced change in tension to change in fluorescence ratio (F340/380) for fetal and adult MCAs. The tension to fluorescence ratio was significantly greater for the adult than fetal MCA. C: vascular tensions (g) for adult and fetal MCAs (symbols same as above) in response to the L-type Ca2+ channel agonist Bay K 8644. Maximum induced tensions were 0.4 ± 0.2 for adult and 1.2 ± 0.2 for fetal vessels. D: fluorescent ratios (F340/380) for adult and fetal MCAs (symbols same as above). Maximum Bay K 8644-induced ratios were 0.07 ± 0.02 and 0.18 ± 0.03 for the adult and fetal vessels, respectively. Inset: ratio of Bay K 8644-induced change in tension to change in fluorescence ratio (F340/380) for fetal and adult MCAs. There was no significant difference between fetal and adult tension to fluorescence ratios.

Figure 4, C and D, shows the contractile tension and fura 2 fluorescence ratio (F340/380) responses of fetal and adult cerebral arteries to the L-type Ca2+ channel agonist Bay K 8644 (10-9-10-6 M). In contrast to stimulation with NE, maximal tensions (g) were significantly greater in the fetus than in the adult (1.2 ± 0.1 vs. 0.5 ± 0.1, respectively; P < 0.01). In addition, Bay K 8644-stimulated maximal [Ca2+]i was also significantly greater in the fetus than in the adult (0.18 ± 0.01 vs. 0.07 ± 0.01, respectively; P < 0.01). Finally, Bay K 8644-stimulated pD2 values for adult and fetal [Ca2+]i were not significantly different, whereas for Bay K 8644-induced tension, the adult pD2 was significantly greater than that of the fetus, 7.7 ± 0.1 vs. 7.1 ± 0.1. The vascular tension and [Ca2+]i of the Bay K 8644 experiments were also calculated as a percent of those obtained during maximum KCl contraction (not shown), but the results were not significantly different.

As shown in the insets of Fig. 4, the ratio of increase in tension to increase in fluorescence ratio (F340/380) for fetal and adult cerebral arteries was not significantly different for Bay K 8644. This contrasts with the somewhat greater tension to [Ca2+]i ratio seen in adult MCA in response to NE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies offer several important observations. First is the striking developmental change in the density of L-type Ca2+ channels in the cerebral arteries and CCAs. Radioligand binding studies revealed a significantly higher L-type Ca2+ channel density in fetal and newborn cerebral arteries and CCAs than in those of the adult (Fig. 1). These results were supported by Western immunoblotting studies (Fig. 2), showing significantly increased L-type Ca2+ channel proteins in fetal vessels. In contrast, radioligand binding studies showed no significant developmental change in L-type Ca2+ channel density in the aorta. Second, there was no significant difference in the KD values of PN200-110 in any of the vessels studied in the radioligand binding study. This suggests no change with development in the type of Ca2+ channel present in VSM. Third, both total and free plasma [Ca2+] were significantly greater in the chronically instrumented fetus than the adult (Fig. 3). Finally, in fetal cerebral arteries, vessel tension and [Ca2+]i were significantly more sensitive to the effects of the L-type Ca2+ channel opener Bay K 8644 compared with the adult vessels (Fig. 4). Nonetheless, the cerebral artery relation of tension to [Ca2+]i in response to Bay K 8644 was not significantly different in the two age groups (Fig. 4D, inset). These results support previous studies from our group of the relative importance of L-type Ca2+ channels in the cerebral arteries of the developing fetus.

L-type Ca2+ channels and development. Several types of voltage-gated Ca2+ channels have been described, e.g., L, N, P/Q, R, and T. These channels reveal a wealth of structural complexity that suggests functional specialization. L-type Ca2+ channels play a key role in the Ca2+ flux of VSM cells. The L-type Ca2+ channels are multimeric proteins consisting of a trans-membrane, pore-forming alpha 1-subunit (190 to 250 KD) in association with several disulfide-linked subunits. The subunits associated with the alpha 1-subunit include a disulfide-linked alpha 2delta -dimer (170 KD), a transmembrane gamma -subunit (33 KD), and an intracellular beta -subunit (55 KD). The alpha 1-subunits confer the characteristic pharmacological and functional properties of these channels, with their function being modulated by the other subunits (1, 5, 13).

L-type Ca2+ channels are blocked by dihydropyridines, phenylakylamines, and/or benz(othi)azepines; each group probably binding a separate but allosterically coupled receptor site (13, 24). Dihydropyridines are allosteric modulators that act on L-type Ca2+ channels as either agonists or antagonists (13). They are believed to access the receptor site, which is located within the pore of the alpha 1-subunit, from the extracellular side of the cell membrane (17). A potent dihydropyridine that binds the L-type Ca2+ channel alpha 1-subunit is PN200-110. Saturation binding of this radiolabeled antagonist is widely used to quantify density of L-type Ca2+ channels (9, 12-14, 16, 25, 31).

Previously, we showed that the dihydropyridine nifedipine is much more effective at blocking NE-stimulated increases in [Ca2+]i and tension of isolated fetal cerebral arteries compared with those of the adult (20). One possible explanation of these results is that developmental differences exist in the affinity of nifedipine for L-type Ca2+ channels. However, the similar KD values observed in the radioligand binding studies of the present study show no significant variation of the channel affinity for PN200-110, making this explanation unlikely. Nonetheless, evidence suggests fetal and adult splice variants of the alpha 1-subunit in the rat heart (7). In addition, various splice forms of L-type Ca2+ channels have been found within VSM cells (1). Sequence analysis and study of the effect of the relative sensitivity of the conductance properties of fetal and adult L-type Ca2+ channels to pharmacological blockers are necessary to rule out the possibility of developmental regulation of the molecular structure of the channels.

Previous work has also shown that fetal and newborn cerebral arteries rely heavily on the uptake of extracellular Ca2+ for contraction (20, 35). In addition, intracellular SR Ca2+ stores are poorly developed in immature vessels (29) and appear to play a minimal role in the contraction of fetal vessels (19). Thus it appears that L-type Ca2+ channels play a much more prominent role in the [Ca2+]i-mediated contraction of fetal cerebral arteries than they do in the adult. Likewise, contraction of bladder smooth muscle from newborn rabbits has been reported to be much more sensitive to the effects of L-type Ca2+ channel than that from adults (34). These results, together with previous work showing that maternally administered nifedipine crosses the ovine placenta, resulting in significant changes in fetal hemodynamics (11), may have implications in the clinical use of L-type Ca2+ channel blockers.

L-type Ca2+ channel density. Another possible mechanism that would facilitate the fetus' increased reliance on extracellular Ca2+ for vessel contraction would be an increase in L-type Ca2+ channel density. This would allow greater Ca2+ influx once the activation voltage had been reached, providing sufficient Ca2+ for contraction despite minimal release of SR Ca2+. Results of the present study support this hypothesis in the cerebral arteries and the CCA. Radioligand binding studies in the present study reveal a two- to threefold greater Bmax in fetal and newborn cerebral arteries and CCAs compared with the adult. Western immunoblotting data show an even greater developmental difference in these vessels. Although other investigators have found similar age-related changes in L-type Ca2+ channel densities in smooth muscle of the rabbit gastrointestinal and urinary tract (15, 33), to our knowledge, this is the first study to demonstrate developmental changes in L-type Ca2+ channel density values in VSM cells.

Other mechanisms might account for the role of L-type Ca2+ channels being greater in the fetus. These include variance in interactions with the intracellular beta -subunit of the L-type Ca2+ channel, which modulates channel activity (1, 31); developmental differences in interaction with the G proteins (8); changes in the molecular structure that alter activation voltages and/or conductance without altering the KD of PN200-110; developmental differences in the organization of the L-type Ca2+ channels on the plasma membrane in relation to the contractile elements; or differences in the phosphorylation effects of modulating protein kinases (5).

An additional finding of the present study is the variation in L-type Ca2+ channel density between the vessel types studied within each age group. In the fetus and newborn, Bmax was significantly greater in both the cerebral arteries and CCA than in the aorta. This may be an indication of the relative importance of these vessels in the regulation of blood flow, the larger aorta being more upstream from the main resistance vessels to which it delivers blood. In the adult, Bmax was significantly greater in the aorta than in the CCAs and cerebral arteries. Here, the difference may be a reflection of the well-developed alpha -adrenergic contraction mechanisms in the resistance vessels resulting in a decreased dependence on L-type Ca2+ channels.

Plasma Ca2+ levels. Another possible factor that would facilitate the fetus' increased reliance on extracellular calcium might be an increased extracellular [Ca2+]. The present study found both ionized and total [Ca2+] to be somewhat elevated in the fetus relative to the adult. These results fit well with previous studies that have provided strong evidence of active Ca2+ transport across the placenta from the fetus to the mother in sheep (3, 27), monkeys (22), and isolated human placenta basal membranes (30). The data are also in agreement with previous studies demonstrating the higher plasma [Ca2+] in the fetus compared with the adult. In acutely instrumented sheep, two studies reported elevated total and free Ca2+ in the fetal plasma compared with the adult (6, 27). Similar findings have been noted in human umbilical cord blood samples at the time of delivery (6, 28). The absolute [Ca2+] values measured in this study also compare well with those from previous studies. This fetal hypercalcemia has been noted to ensure that the fetus has enough Ca2+ to maintain bone growth (26). Although the relative difference between fetal and adult plasma [Ca2+] is minimal compared with the large difference between extracellular and intracellular [Ca2+], recent evidence of the fetus' increased reliance on extracellular Ca2+ for vessel contraction attaches additional importance to the role of active placental Ca2+ transport. The evidence also emphasizes the importance of factors controlling the ratio of ionized to protein-bound Ca2+ in the fetal plasma, which would be important in determining the amount of Ca2+ available for vessel contraction, a topic requiring further study.

Bay K 8644 studies. Previous work from our group showed that L-type Ca2+ channel blockers have a much stronger inhibition of contraction on fetal vessels than on adult vessels (20). In the present study, we observed that fetal vessels are significantly more sensitive to the effect of the L-type Ca2+ channel agonist Bay K 8644. Both contractile tension and [Ca2+]i showed this effect. In fetal cerebral arteries, the maximal contractile tension was essentially the same whether the contraction was stimulated by NE or Bay K 8644. This supports the idea that, while the NE-stimulated response is alpha -adrenergic-receptor (alpha 1-AR) mediated, the activation of L-type Ca2+ channels is likely to be an important component of the signal transduction pathway. The fact that the maximal contraction of adult vessels is significantly less in response to Bay K 8644 compared with that observed with NE underscores the importance of the intracellular SR Ca2+ stores in the contraction of adult vessels, the L-type Ca2+ channels not providing enough Ca2+ flux to produce even half as much contraction as the alpha 1-AR-mediated pathway. These findings are in agreement with previous work by van Breemen and Siegel (31) who demonstrated the effect of NE on the release of Ca2+ from the SR in mature rabbit aortae.

Perspectives

The current study provides further evidence to support the idea that fetal cerebral vessels rely to a greater extent on extracellular sources of calcium for contraction than do those vessels in the adult and demonstrates an increased number of L-type Ca2+ channels to mediate this. It also demonstrates an increased availability of extracellular Ca2+ for contraction in the fetus. Further work is needed to rule out a developmental change in the molecular structure of the L-type Ca2+ channel, including protein sequencing and patch-clamping to determine possible developmental differences in activation voltages or conductances. Renewed consideration of the role that Ca2+-binding proteins play in the control of free Ca2+ in the plasma of the fetus may also provide valuable information. Overall, the present studies add to the concept of the role of extracellular Ca2+ in fetal vessel contractility and to the importance of developmental differences in vascular signal transduction mechanisms.


    ACKNOWLEDGEMENTS

We thank B. Kreutzer for preparing the manuscript.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HD/HL-03807 and PO1-HD-31226 to L. D. Longo.

Address for reprint requests and other correspondence: L. D. Longo, Center for Perinatal Biology, Loma Linda Univ., School of Medicine, Loma Linda, CA 92350 (E-mail: llongo{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 6 June 2001; accepted in final form 19 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 282(1):R131-R138
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