INTRODUCTION

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INTRACELLULAR CALCIUM ([Ca²⁺]_i) has a central role in the physiology and pathophysiology of arterial smooth muscle by regulating contractility and gene expression. Elevated [Ca²⁺]_i has been implicated in both contractile and phenotypic changes of arteries in hypertension (7, 13, 16, 20, 24, 25). Consistent with elevated [Ca²⁺]_i, L-type voltage-dependent Ca²⁺ channel (VDCC) currents have been reported to be enhanced in vascular smooth muscle during hypertension through both upregulation of the channel itself (29) and through membrane potential depolarization (10). An alteration in the membrane potential of the vascular smooth muscle during hypertension may reflect changes in the permeability of K⁺ in these cells. Decreased expression of voltage-dependent K⁺ (K_v) channels has been reported in renal arteries from hypertensive rats (16) and in pulmonary arteries from humans with primary pulmonary hypertension (30). Such a decrease in K_v currents would promote membrane potential depolarization. Conversely, Ca²⁺-sensitive K⁺ (K_Ca) channel activity is enhanced in vascular smooth muscle from hypertensive animals in an attempt to compensate for the elevation in [Ca²⁺]_i (14, 15, 21). With respect to the cerebral vasculature, the effects of hypertension on K⁺ channel activity or [Ca²⁺]_i are poorly understood. Consistent with findings from other vascular beds (7, 16), the membrane potential of cerebral arteries from spontaneously hypertensive rats has been reported to be more depolarized (9).

Although functional and structural changes in smooth muscle during hypertension reflect, in part, altered Ca²⁺ handling and gene expression, the relationship between [Ca²⁺]_i and gene expression is not known. Recently, Cartin et al. (4) explored the Ca²⁺ dependence of gene expression in arterial smooth muscle of intact cerebral arteries of the mouse. In this preparation, a marked increase in the expression of the immediate early gene c-fos was observed when Ca²⁺ influx through VDCCs was elevated by membrane potential depolarization. Furthermore, the signaling cascade linking an increase in [Ca²⁺]_i to enhanced gene expression in cerebral arterial smooth muscle appears to involve activation and phosphorylation of the transcription factor cAMP response element binding protein (CREB) by a Ca²⁺-calmodulin-dependent protein kinase (CaM kinase). A similar pathway linking Ca²⁺ influx to increased c-fos expression via CaM kinase-dependent phosphorylation of CREB has previously been identified in hippocampal neurons (1, 5).

Increased expression of c-fos may also be involved in the cascade of events leading to phenotypic changes in vascular smooth muscle that occur during hypertension. Exposure to hypertrophic and/or proliferative stimuli such as platelet-derived growth factor (PDGF), thrombin, or angiotensin II caused an increase in c-fos levels in cultured aortic (28) and pulmonary arterial smooth muscle cells (24, 27). In cultured vascular smooth muscle, elevations in [Ca²⁺]_i may play an important role in enhanced c-fos expression. Increased c-fos expression has been reported to occur in these cultured myocytes in response to mobilization of Ca²⁺ from intracellular stores (17, 24, 28) or via Ca²⁺ influx through dihydropyridine-sensitive L-type VDCCs (13). However, caution should be exercised in extrapolating studies of cultured vascular smooth muscle (synthetic phenotype) to native vascular smooth muscle (contractile phenotype).

The goal of this study was to examine the role of [Ca²⁺]_i in mediating changes in gene expression of cerebral arterial smooth muscle obtained from a rat genetic model of salt-sensitive hypertension. Our results indicate that in hypertension, the membrane potential is depolarized, which likely reflects a decrease in K_v current density. [Ca²⁺]_i is elevated in hypertension, which appears to result from an elevation of Ca²⁺ influx through VDCCs. This increase in [Ca²⁺]_i leads to an activation of CREB and elevation of gene expression (c-fos).

	METHODS

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Dahl salt-sensitive (SS, "hypertensive") and Dahl salt-resistant (SR, "normotensive") rats 6 wk of age were fed an 8% NaCl diet for 6 wk. Mean arterial blood pressure measurements were taken in these animals using the tail-cuff method. Animals were euthanized by exsanguination while under deep pentobarbital anesthesia (intraperitoneal; 150 mg/kg). Cerebral arteries were dissected in cold (4°C) oxygenated (95% O₂-5% CO₂) physiological saline solution (PSS) of the following composition (in mM): 118.5 NaCl, 4.7 KCl, 24 NaHCO₃, 1.18 KH₂PO₄, 2.5 CaCl₂, 1.2 MgCl₂, 0.023 EDTA, and 11 glucose. Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (Publication 85-23) following protocols approved by the Institutional Animal Use and Care Committee of the University of Vermont.

Membrane potential measurements. Membrane potential measurements were made in arterial myocytes of isolated posterior cerebral artery segments pressurized to 10 mmHg and continuously superfused with oxygenated PSS (37°C). Conventional intracellular microelectrodes filled with 0.5 M KCl were used to impale myocytes from the adventitial surface of arteries. Acceptance of membrane potential recordings was based on previously described criteria (3).

K+ current recordings. Smooth muscle cells were enzymatically isolated from cerebral arteries (22), and whole cell K⁺ currents were measured in these freshly isolated myocytes using the conventional whole cell configuration of the patch-clamp technique. Patch pipettes were filled with a solution containing (in mM) 87 potassium aspartate, 20 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 EGTA, and 25 KOH (pH 7.2). Free [Ca²⁺] in the pipette solution was calculated to be ~100 nM. Recordings were made in an extracellular solution containing (in mM) 134 NaCl, 6 KCl, 1 MgCl₂, 0.1 CaCl₂, 10 glucose, and 10 HEPES (pH 7.4). To compensate for differences in cell size, membrane K⁺ currents are expressed relative to cell capacitance (pA/pF).

Arterial wall Ca²+ measurements. Cannulated arteries were loaded with the ratiometric Ca²⁺-sensitive dye fura-2 acetoxymethyl ester (AM) (2 µM) and held at 10 mmHg while continuously superfused with oxygenated PSS (37°C). Arterial [Ca²⁺]_i was calculated in fura-2-loaded arteries as described previously (12).

Immunofluorescence. Cerebral arteries were treated with test stimuli for 30 min at 37°C. When indicated, isolated arteries were incubated in 100 nM nisoldipine for 15 min before incubation with 60 mM K⁺, then flash-frozen, and embedded. Arterial cross sections (10 µm thick) were fixed and treated with primary antibody [rabbit anti-phosphorylated (P-CREB), 1:250 dilution in 0.2% Triton-X/2% bovine serum albumin-phosphate-buffered saline, New England Biolabs; Beverly, MA] followed by secondary antibody (Cy3 or Cy5-anti-rabbit IgG, 1:500 dilution in 2% bovine serum albumin-phosphate-buffered saline) (4, 26). Control experiments using either primary antibody only or secondary antibody only (on different slices) revealed virtually no nonspecific staining (data not shown). In addition, the phosphopeptide (1 mg/ml) used to generate the anti-P-CREB antibody was obtained from Cell Signaling Technology (Beverly, MA). No positive P-CREB staining was evident in arteries stimulated with high K⁺ (n = 3 artery sections from each of 3 animals, data not shown) when the anti-P-CREB antibody was incubated for 2 h with the phospho-peptide (1:250 dilution) before staining. Sections were also stained with the cyanine dye YOYO-1 (1:5,000 in PBS) to identify nuclei. Arterial sections contained between 20 and 50 nuclei, and 6 sections per treatment were analyzed for each animal using standardized conditions within Adobe Photoshop.

RNA isolation and RT-PCR. Arteries were treated in a manner similar to that described for immunofluorescence. Total RNA was extracted using TRIzol reagent and quantified using a spectrophotometer. First-strand cDNA was synthesized using Superscript II RNase H-Reverse Transcriptase. To perform RT-PCR, cDNA was amplified using the following sets of primers: c-fos (GenBank Accession no. X06769) sense, nucleotides 1131-1152, and antisense 1426-1446; GAPDH (GenBank Accession no. M17701) sense nucleotides 586-606, and antisense 1017-1037. RT-PCR was performed under the following conditions: 94°C, 1 min; 55°C, 1 min; and 72°C, 2 min for 35 cycles, followed by an incubation at 72°C for 10 min. Experiments have confirmed that the number of PCR cycles (35) used in our study is within the linear amplification range of this assay (n = 3, data not shown). For each animal, reactions with c-fos and GAPDH primers were run next to each other in the thermal cycler and thus exposed to the same PCR cycle. To also reduce variability between tubes, a PCR master mix was prepared containing cDNA, 10 mM deoxynucleotide triphosphates (2.5 mM each), 10 µl of 5× PCR buffer (containing 7.5 mM MgCl₂, pH 8.5), and 5 units of Taq Polymerase (5 U/µl) per reaction. Sense and antisense primers (0.25 µg each) were then added to the individual reaction tubes. The final concentration of each reaction was adjusted to 50 µl using sterile water. PCR products were separated by agarose gel electrophoresis and quantified using Adobe Photoshop Software. Equal amounts of cDNA were loaded on the gel, and c-fos DNA concentration was normalized to GAPDH expression. All results are expressed as means ± SE.

	RESULTS

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Cerebral arteries from hypertensive rats are depolarized compared with normotensive animals. Mean arterial pressure was elevated to 183.7 ± 2.7 mmHg (n = 21) in Dahl SS rats, whereas Dahl SR rats remained normotensive (112.4 ± 3.1 mmHg, n = 12) following a period of 6 wk on high-salt (containing 8% NaCl) diet. To investigate whether this model of hypertension is associated with altered electrical properties of arterial myocytes within the cerebral vasculature, intracellular microelectrodes were used to measure the membrane potential of smooth muscle cells in intact isolated cerebral arteries. As illustrated in Fig. 1A and summarized in Fig. 1B, the membrane potential of cerebral artery myocytes from hypertensive rats (36.8 ± 0.8 mV, n = 16 cells from 4 animals) was depolarized compared with similar cells from normotensive animals (49.7 ± 1.2 mV, n = 12 cells from 3 animals).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Cerebral arteries from hypertensive rats are depolarized compared with normotensive animals. A: conventional intracellular microelectrode recordings of membrane potential measurements in myocytes of intact cerebral artery segments from normotensive (left) and hypertensive (right) rats. B: summary of the membrane potential of myocytes from normotensive (n = 12 cells from 3 animals) and hypertensive (n = 16 cells from 4 animals) cerebral arteries. *P < 0.01

Voltage-dependent K+ channel current density is decreased in smooth muscle cells from cerebral arteries of hypertensive rats. The observed membrane depolarization of cerebral arterial smooth muscle from hypertensive animals could reflect either enhanced depolarizing stimuli, decreased hyperpolarizing stimuli, or both. To determine whether K⁺ channel currents were altered in cerebral arteries of hypertensive animals, outward K⁺ currents were directly measured in isolated myocytes using the patch-clamp technique. Mean series resistance was 11.0 ± 1.4 M (n = 21) and 8.5 ± 0.6 M (n = 21) in cells from normotensive and hypertensive animals, respectively. Cell capacitance was 15.5 ± 0.6 pF (n = 21) in cells from normotensive and 9.2 ± 0.4 pF (n = 21) in cells from hypertensive animals. Membrane K⁺ currents (in 6 mM external K⁺) were elicited by a series of 10-mV depolarizing steps (60 to +50 mV) from a holding potential of 70 mV (Fig. 2A). Steady-state outward current was significantly less in cerebral artery myocytes isolated from hypertensive animals (15.2 ± 1.7 pA/pF at +50 mV, n = 21) compared with similar cells from normotensive animals (34.8 ± 5.6 pA/pF at +50 mV, n = 21, P < 0.01, Fig. 2B). Peak outward currents (i.e., the maximum level of current during a given voltage step) were also significantly less in cerebral arteries from hypertensive (23.2 ± 3.0 pA/pF at + 50 mV, n = 21) compared with cells from normotensive rats (39.9 ± 6.2 pA/pF at + 50 mV, n = 21, P < 0.05). To determine the nature of the outward currents in these cells, membrane currents were also measured in the presence of iberiotoxin (IBTX, 100 nM, a selective inhibitor K_Ca channels), and IBTX plus 4-aminopyridine (4-AP, 10 mM, an inhibitor of some types of K_v channels) (Fig. 3A). IBTX-sensitive currents were not significantly different in cerebral artery myocytes from normotensive compared with hypertensive animals (Fig. 3B). However, in the presence of IBTX, both the 4-AP-sensitive and -insensitive currents were significantly less in cells isolated from hypertensive animals at depolarized membrane potentials (Fig. 3, C and D). These data are consistent with the idea that decreased K_v channel conductance leads to the depolarized membrane potential observed in cerebral arteries of hypertensive rats.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Voltage-dependent K+ channel currents are decreased in cerebral artery myocytes from hypertensive animals. A: outward membrane currents elicited by a series of 10-mV depolarizing steps (60 to +50 mV) from a holding potential of 70 mV in normotensive (left) and hypertensive (right) cerebral artery myocytes. Cell capacitance was 17.5 and 8.5 pF in the myocyte isolated from a normotensive and hypertensive animal, respectively. B: summary of the steady-state current-voltage relationship of voltage-dependent K+ channel currents in myocytes obtained from normotensive (squares, n = 21) and hypertensive (circles, n = 21) cerebral arteries. *P < 0.01.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   4-Aminopyridine (4-AP)-sensitive and -insensitive voltage-dependent K+ channel currents are decreased in cerebral artery myocytes from hypertensive animals. A: relative contribution of iberiotoxin (IBTX)- and 4-AP-sensitive components of outward membrane K+ currents in cerebral artery myocytes isolated from normotensive (n = 6) and hypertensive animals (n = 6). B-D: current density of IBTX-sensitive (B), 4-AP-sensitive (C), and IBTX- and 4-AP-insensitive (D) currents in cerebral artery myocytes isolated from normotensive (n = 6) and hypertensive (n = 6) animals. *P < 0.05.

Elevation of arterial wall Ca²+ in cerebral arteries of hypertensive rats. The membrane potential depolarization observed in cerebral arteries from hypertensive animals should lead to an elevation of [Ca²⁺]_i in the smooth muscle within the arterial wall due to increased Ca²⁺ influx through VDCCs (12). In arteries from normotensive animals, arterial wall Ca²⁺ was 134.9 ± 15.7 nM (n = 4), which is similar to measurements of arterial wall Ca²⁺ obtained from other strains of normotensive rats under similar conditions (12). However, in cerebral arteries from hypertensive rats, arterial wall Ca²⁺ was elevated to 224.4 ± 37.9 nM (n = 5). The organic inhibitor of VDCCs, diltiazem (50 µM), reduced [Ca²⁺]_i in cerebral arteries from hypertensive and normotensive animals to similar levels (118.6 ± 16.8 nM, n = 4 and 111.2 ± 9.8 nM, n = 6, respectively), indicating that the increase in arterial wall Ca²⁺ in arteries from hypertensive animals was due to enhanced Ca²⁺ entry. Furthermore, membrane potential depolarization, by elevating extracellular K⁺ from 6 to 30 mM, increased [Ca²⁺]_i approximately 1.6-fold in cerebral arteries from normotensive animals to similar [Ca²⁺]_i levels observed in hypertensive arteries under resting (6 mM extracellular K⁺) conditions.

Activation of the transcription factor CREB in hypertensive cerebral arteries. An elevation of [Ca²⁺]_i by membrane potential depolarization has recently been shown to activate the transcripton factor CREB, by increasing the levels of P-CREB (4). Because smooth muscle cells in hypertensive cerebral arteries are depolarized and exhibit elevated levels of [Ca²⁺]_i, we hypothesized that levels of P-CREB should also be increased in cerebral arteries from hypertensive rats. Cross sections of cerebral arteries (10 µm thick) were analyzed for P-CREB by immunostaining with an antibody specific for CREB phosphorylated on Ser¹³³. Sections were also stained with the cyanine dye YOYO-1 to identify nuclei. Cerebral arteries from hypertensive rats exhibited a significant increase in the number of nuclei staining positive for P-CREB (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Elevated levels of phosphorylated cAMP response element binding protein (P-CREB) in cerebral arteries from hypertensive rats are reversed by the voltage-dependent Ca2+ channel (VDCC) inhibitor nisoldipine (Nis). Arterial cross sections were stained with anti-P-CREB antibody and the nuclear dye YOYO-1. Immunofluorescent images of cerebral arteries from normotensive rats (A) and hypertensive rats (B) are shown. C: summary of effects of membrane depolarization and nisoldipine on levels of P-CREB. Arterial cross sections were evaluated for the percentage of cells staining positive for nuclear P-CREB (n = 3 arteries, 6 sections per artery). PSS, physiological saline solution. *P < 0.05, Student-Newman-Keuls.

Ca²+-dependent increase in c-fos expression in hypertensive cerebral arteries. Activation of CREB by [Ca²⁺]_i in mouse cerebral arteries is associated with an increase in expression of the immediate early gene c-fos (4). To examine whether the increased phosphorylation of CREB during hypertension also correlates with increased c-fos expression, RT-PCR was performed on RNA extracted from intact cerebral arteries from normotensive and hypertensive rats (Fig. 5). Treatment with the Ca²⁺ channel blocker nisoldipine (100 nM) significantly reduced the c-fos-to-GAPDH ratio in cerebral arteries from hypertensive, but not normotensive, arteries. Whereas membrane depolarization with 60 mM KCl increased the c-fos-to-GAPDH ratio sevenfold in arteries from normotensive animals, this maneuver did not increase c-fos expression in arteries from hypertensive animals. Although RT-PCR measurements performed in this study are qualitative in nature, these results suggest that Ca²⁺ influx through VDCC leads to increased expression of c-fos in cerebral arteries from hypertensive rats.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of membrane depolarization and VDCC inhibition on c-fos levels in cerebral arteries from normotensive and hypertensive rats. RT-PCR was performed on RNA extracted from intact cerebral arteries from normotensive (A) and hypertensive (B) rats. Quantitation of c-fos mRNA levels is expressed relative to GAPDH mRNA, as determined by pixel intensity of scanned gels (n = 3 animals for each group). *P < 0.01, Student-Newman-Keuls.

	DISCUSSION

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Hypertension is a complex disease that can arise from a multitude of underlying genetic and environmental factors. Many, if not all, forms of hypertension result in a number of direct effects on the vasculature that include increased contractility, vascular smooth muscle proliferation, and remodeling of the arterial wall. An increase in [Ca²⁺]_i in vascular smooth muscle could play an important role in many of these pathological changes. To this extent, we have investigated whether [Ca²⁺]_i is altered in cerebral arteries during hypertension using a genetic rat model of salt-sensitive hypertension. We report that in the Dahl model of salt-sensitive hypertension, [Ca²⁺]_i is elevated in the smooth muscle of cerebral arteries due to membrane potential depolarization, likely reflecting a decrease in K_v channel conductance. Furthermore, we provide evidence that this depolarization-induced increase in [Ca²⁺]_i is due to increased Ca²⁺ entry through VDCCs and leads to enhanced activation of the transcription factor CREB and increased expression of the immediate early gene c-fos. This study provides the first information linking altered Ca²⁺ handling to changes in gene expression in cerebral arteries during hypertension.

Membrane depolarization and elevated [Ca²+]_i in cerebral artery myocytes with hypertension: role of K_v channels. In cerebral arterial smooth muscle, [Ca²⁺]_i is regulated by Ca²⁺ influx through VDCCs (12, 19). The activity of K⁺ channels is a key determinant of the membrane potential of cerebral vascular smooth muscle. At least two types of K⁺ channels, voltage-dependent (K_v, or delayed rectifier), and large-conductance Ca²⁺-activated K⁺ channels play a key negative-feedback role to limit Ca²⁺ entry and pressure-induced constrictions in the cerebral vasculature (2, 6, 11, 18). We have found the K_v channel current density was markedly suppressed in cerebral artery myocytes from hypertensive rats. Our findings are also consistent with reports from other investigators describing decreased K_v currents in other rat models of hypertension (16) and human patients with primary pulmonary hypertension (30). It is also interesting to note that parathyroid hypertensive factor, a substance correlated with low renin, salt-sensitive hypertension in humans, also inhibits K_v currents in cultured vascular smooth muscle cells (23). The mechanism underlying the decrease in K_v channel activity remains to be elucidated.

Activation of CREB and c-fos expression in hypertension. We found that the levels of P-CREB-positive nuclei and c-fos expression were elevated in cerebral arteries from hypertensive rats. Membrane potential depolarization of normotensive arteries mimicked the effects of hypertension. In both cases, inhibition of VDCCs decreased the levels of P-CREB and c-fos. These results are consistent with our measurements indicating that smooth muscle cells of cerebral arteries from hypertensive animals exhibit a more depolarized membrane potential and elevated [Ca²⁺]_i due to increased Ca²⁺ influx through VDCCs. Unlike arteries from normotensive animals, elevating external K⁺ from 6 to 60 mM did not significantly increase levels of P-CREB or c-fos in cerebral arteries from hypertensive animals. A likely explanation for the inability of elevated K⁺ to increase P-CREB levels or c-fos expression in hypertensive arteries is that the membrane potential of these arteries under resting conditions (low pressure, 6 mM external K⁺) is near the equilibrium potential for K⁺ (E_K) predicted for 30 mM external K⁺ (E_K 40 mV) or 60 mM external K⁺ (E_K 22 mV). The relationship between membrane potential (or [Ca²⁺]_i) and activation of the transcription factor CREB in arterial smooth muscle appears relatively steep in arteries from normotensive animals, because elevating extracellular K⁺ from 6 mM to either 30 or 60 mM caused a similar increase in the phosphorylation of CREB to its activated form (P-CREB). As a cautionary note, the experiments described in the present study were performed on myocytes from either unpressurized cerebral arteries (P-CREB, c-fos, and patch-clamp studies) or arteries subjected to very low (10 mmHg) intravascular pressure (membrane potential and arterial wall Ca²⁺ measurements). Future studies are necessary to define the relationship between Ca²⁺-dependent gene expression and intravascular pressure with respect to hypertension.