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(Circulation Research. 2004;94:e97.)
© 2004 American Heart Association, Inc.

UltraRapid Communications

High Blood Pressure Upregulates Arterial L-Type Ca²⁺ Channels

Is Membrane Depolarization the Signal?

Aleksandra Pesic, Jane A. Madden, Miodrag Pesic, Nancy J. Rusch

From the Department of Pharmacology and Toxicology (A.P., M.P., N.J.R.), The Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wis; and the Department of Neurology (J.A.M.), Medical College of Wisconsin and Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wis.

Correspondence to Nancy J. Rusch, PhD, Prof, Department of Pharmacology and Toxicology Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail nrusch{at}mcw.edu

	Abstract

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Long-lasting Ca²⁺ (Ca_L) channels of the Ca_v1.2 gene family contribute to the pathogenesis of abnormal arterial tone in hypertension. The physiological stimulus that enhances Ca_L channel current in the vascular smooth muscle cells (VSMCs) remains unknown. The present study investigated if high blood pressure triggers an upregulation of vascular Ca_L channel protein. Rat aortae were banded between the origins of the left renal (LR) and right renal (RR) arteries to selectively elevate blood pressure in the proximal RR arteries. After 2 days, the immunoreactivity on Western blots corresponding to the pore-forming _1C subunit of the Ca_L channel was increased 3.25-fold in RR compared with LR arteries. This finding persisted at 28 days and was associated with abnormal Ca²⁺-dependent tone and higher Ca_L currents in the VSMCs exposed to high pressure. Based on microelectrode studies indicating that RR arteries were depolarized compared with LR arteries, further studies examined if membrane depolarization, an inherent response of VSMCs to high blood pressure, increased _1C expression. Isolated rat renal arteries were cultured for 2 days in low K⁺ (4 mmol/L) or depolarizing high K⁺ (30 mmol/L) media. Arteries preconditioned in high K⁺ showed a 5.47-fold increase in _1C expression, enhanced Ca_L channel current, and elevated Ca²⁺-dependent tone. These findings provide the first direct evidence that high blood pressure upregulates the Ca_L channel _1C subunit in VSMCs in vivo and suggest that membrane depolarization is a potential signal involved in this interaction that may contribute to the development of abnormal vascular tone.

Key Words: calcium channels • _1C subunit • membrane potential • vascular smooth muscle • hypertension

	Introduction

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Voltage-gated calcium influx through L-type Ca²⁺ (Ca_L) channels of the Ca_v1.2 gene family regulates the diameters of small arteries and arterioles, and maintains the myogenic tone of resistance vessels.^1,2 At the protein level in vascular smooth muscle cells (VSMCs), the pore of the Ca_L channel is formed by the _1C subunit that represents a splice variant (_1C–b) of the cardiac _1C-a gene.³ Ultimately, _1C is presumed to assemble into heteromultimers with ancillary ß and ₂ subunits, which determine the properties and expression level of the Ca_L channel at the cell surface.⁴ However, although the Ca_L channel is a major determinant of vascular reactivity and a major target of vasodilator therapies, the key physiological factors that regulate its expression level in the arterial circulation have not been identified.

In this regard, our laboratory recently reported that hypertension, a disease characterized by abnormal vascular tone, is associated with an upregulation of the _1C subunit in systemic arteries of spontaneously hypertensive rats (SHR).⁵ This finding may explain, at least in part, earlier observations that Ca_L current is increased in VSMCs from several SHR arteries^6–9 and from the cerebral arteries of rats with renal and diet-induced hypertension.^10,11 However, the mechanism of the elevated Ca_L current in hypertension is not readily apparent.¹² In genetically hypertensive rats, the higher expression levels of vascular _1C may be an epiphenomenon of the different genetic backgrounds of the hypertensive and normotensive animals, rather than a participant in the pathogenesis of hypertension.^5,12 Similarly, the neuroendocrine profiles of rats with induced forms of hypertension differ greatly from control rats of the same strain,¹³ which complicates the identification of pressure versus other physiological factors as the source of _1C upregulation. Nonetheless, the recurrent finding of increased Ca_L current in VSMCs from several rat models of hypertension implies a unique relationship between blood pressure and the availability of Ca_L channels. One intriguing possibility is that high blood pressure per se promotes the expression of the Ca_L channel protein in the arterial circulation. Indeed, VSMCs in small arteries profoundly depolarize in response to high perfusion pressures,^1,14,15 a signal that has been linked to the appearance of functional Ca_L channels in cultured chick myotubes exposed to high K⁺ solutions.¹⁶

The present study was designed to test the hypothesis that high blood pressure induces the expression of arterial Ca_L channels in vivo. To examine this issue, we banded the rat aorta between the origins of the right renal (RR) and left renal (LR) arteries to selectively expose the VSMCs of the proximal RR arteries to high blood pressure.¹⁷ As early as 2 days after banding, the RR arteries demonstrated an increased expression of the Ca_L channel _1C subunit compared with the LR arteries exposed to lower blood pressure. Furthermore, simply preconditioning isolated rat renal arteries in depolarizing media for 2 days increased _1C expression and the level of functional Ca_L channels. Our findings suggest a new paradigm for understanding the pathogenesis of hypertension, whereby the depolarizing influence of high blood pressure may trigger the upregulation of arterial Ca_L channels to further promote the development of abnormal vascular tone.

	Materials and Methods

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Animals
All procedures involving animals were approved by the institutional Animal Care and Use Committee. To perform interrenal aortic banding, Sprague-Dawley rats obtained from Harlan Laboratories (Madison, Wis) at 10 weeks of age were deeply anesthetized (1:10 vol:vol of ketajet-acepromazine, IM), a midline abdominal incision performed, and the aorta was visualized.¹⁷ A 22-gauge needle was placed adjacent to the abdominal aorta, and a strand of 3-0 silk suture was jointly tightened around the aorta and needle between the origin of the LR and RR arteries. The ligature was secured and the needle removed to permit the partial return of blood flow through the banded aorta. Sham-operated rats were exposed to the same surgery, but the aortic ligature was not tightened. After recovery, all rats were allowed free access to food and water. On the day of studies, rats were anesthetized again and the kidneys removed to obtain the renal arteries.

Vessel Culture
To determine the effect of membrane depolarization on vascular Ca_L channel expression, small renal arteries were microdissected from the renal parenchyma of normal Sprague-Dawley rats, and cultured for 2 days in control DMEM (4 mmol/L K⁺) or depolarizing DMEM containing 30 mmol/L K⁺. Both culture media contained a low concentration of 0.1% fetal bovine serum to discourage VSMC dedifferentiation.

Microelectrode Recordings
To determine if resting membrane potential (E_m) differed between LR and RR arteries of aortic-banded rats, resting E_m was recorded by glass microelectrodes in cannulated LR and RR arteries perfused at their respective in vivo blood pressure levels.¹⁸

Analysis of Ca_L Channel Expression and Current
Membrane proteins were prepared from the intraparenchymal renal arteries of the left and right kidneys. The expression level of the pore-forming _1C subunit of the Ca_L channel was compared by Western blotting using a polyclonal antibody characterized in detail earlier.⁵ For patch-clamp studies, VSMCs from renal arcuate arteries were enzymatically isolated and studied within 6 hours.^5,18 Whole-cell Ca²⁺ channel current densities were assessed using standard pulse protocols and a patch-clamp station described previously.¹⁹ Experiments were performed at 25°C using 10 mmol/L barium chloride as the charge carrier to limit current rundown. The composition of the pipette solution was (in mmol/L) cesium glutamate 145, MgCl₂ 1, HEPES 10, EGTA 10, and Na₂ATP 3 (pH 7.2). The bath solution contained (in mmol/L) BaCl₂ 10, TEA 135, MgCl₂ 1, HEPES 10, and glucose 10 (pH=7.4). In a subset of cells, 1 µmol/L nifedipine (Sigma) was used to verify the identity of Ca_L channel currents.

Vascular Reactivity Assays
To evaluate the contribution of Ca_L channels to vascular tone, concentration-dependent responses to the dihydropyridine agonist, Bay K8644 (Sigma), were measured in isolated renal arterial rings mounted for tension-recording.⁵ The level of nifedipine-sensitive basal tone mediated by the spontaneous opening of Ca_L channels also was assessed in similar arterial rings.⁵

Statistics
Unpaired t tests were used to evaluate significance of single point comparisons. Comparison of multiple points generated in patch-clamp and vessel reactivity studies were tested by two-way ANOVA with repeated measures, followed by a Duncan’s multiple range test. Significance was accepted at P<0.05. Sample sizes are indicated in the figure legends.

	Results

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Pressure Upregulates _1C in Arteries In Vivo
The aortae of rats were banded between the origins of the LR and RR arteries to selectively elevate blood pressure in the RR circulation (Figure 1A). Systolic pressures distal and proximal to the banded site were measured in anesthetized rats using femoral and carotid catheters at 2, 7, and 28 days after banding. In a total of 82 rats, systolic blood pressure below the banded site and perfusing the LR arteries remained low after banding, averaging 63±19 (n=11), 80±24 (n=15), and 86±19 mm Hg (n=56) at 2, 7, and 28 days, respectively. In contrast, systolic blood pressure proximal to the banded site that perfused the RR arteries in the same animals was elevated to 143±26, 157±15, and 195±30 mm Hg at 2, 7, and 28 days after banding, respectively (Figure 1B). Thus, the RR arteries of aortic-banded rats were exposed to similar genetic and humoral influences, but persistently higher levels of blood pressure than the LR arteries of the same animals.

View larger version (52K): [in this window] [in a new window]   Figure 1. A, Rat model of interrenal aortic banding. Blood pressure is selectively increased in the right renal circulation. B, Profile of systolic blood pressures in the left renal (LR) and right renal (RR) artery estimated by femoral and carotid catheters, respectively, at 2, 7, and 28 days after banding. *Significant difference (P<0.05) between LR and RR values. C, Western blot comparing the 200- to 240-kDa doublet immunoreactive band associated with the 1C short and long forms between LR and RR arteries of 28-day banded rats. Expression of the -actin internal standard (45 kDa) was unchanged. Proteins were pooled from 4 animals. C, inset, Comparison of the 1C immunoreactive bands between LR and RR arteries of a single rat. D, Expression of 1C was not increased in RR compared with LR arteries of sham-operated rats at 28 days. Proteins were pooled from 3 rats. E and F, Comparison of 1C expression between LR and RR arteries of single rats at 2 days (E) and 7 days (F) after banding, respectively. G, At 2 and 7 days, 1C upregulation was not detected in a subset of rats.

Based on earlier studies demonstrating an increased availability of vascular Ca_L channels in the chronic stage of hypertension,^6–11 Western blots were utilized to examine the effect of blood pressure elevation on the expression of Ca_L channel _1C subunits at 28 days after banding. These studies demonstrated a profound upregulation of _1C in the RR arteries exposed to high pressure, compared with the LR arteries distal to the banded site (Figure 1C). In contrast, the expression of an internal standard, smooth muscle -actin, was similar between LR and RR arteries. Although arteries were generally pooled from several rats to provide higher protein yields, an enhanced expression of _1C in RR arteries also was detected in vascular proteins from single rats (Figure 1C, inset). In 6 Western blots using different protein batches, the immunoreactivity of the doublet bands at 200 and 240 kDa corresponding to the short and full-length forms of _1C, respectively, was increased 1.76-fold in RR compared with LR arteries. Although the expression of the _1C long form was consistently but mildly elevated by an average of 1.30-fold, expression of the _1C short form, which exists as a C-terminus truncation of the full-length protein, was increased by 2.52-fold in RR compared with LR arteries. Notably, the elevated levels of _1C in the RR arteries could not be attributed to a preexisting difference in protein expression between the LR and RR circulations, or to components of the banding surgery unrelated to the induction of high blood pressure. Instead, five Western blots indicated that the natural expression level of _1C in RR arteries was slightly (16%) less than in LR arteries of sham animals (Figures 1D and 2). In sham-operated animals, a ligature was tied around the aorta but not tightened so that systolic blood pressure remained normal above (129±25 mm Hg) and below (124±22 mm Hg) the banded site at 28 days.

View larger version (30K): [in this window] [in a new window]   Figure 2. Bar graph plotting the ratio of immunoreactivity corresponding to 1C in Western blots loaded with right renal (RR) or left renal (LR) proteins obtained from sham-operated rats or from rats banded for 2, 7, or 28 days to selectively increase blood pressure in the RR arteries in vivo (n=5, 11, 15, and 6 blots, respectively). *Ratio of RR/LR immunoreactivity was significantly different (P<0.05) than unity.

New studies were performed at 2 and 7 days after banding to determine the rapidity of the _1C response to high blood pressure. To ensure an adequate blood pressure stimulus, only renal arteries from animals that demonstrated a minimum pressure difference of 40 mm Hg across the banded site were used. In these animals, _1C expression was clearly increased in RR compared with LR arteries from 8 of 11 rats (73%) at 2 days after banding (Figure 1E). Similarly, the RR arteries from 11 of 15 rats (73%) showed an increased expression of _1C at 7 days (Figure 1F). In the RR arteries of the remaining animals (27%), no increase in _1C was detected at these earlier time limits (Figure 1G). Including all animals used for Western blot screening (n=59), the total immunoreactivity corresponding to the _1C protein was increased by 3.25-fold (day 2), 2.76-fold (day 7), and 1.76-fold (day 28) in RR arteries exposed to elevated blood pressure, inferring a rapid effect of high pressure on Ca_L channel expression that peaked at 2 days but persisted at 28 days after banding (Figure 2).

Pressure Increases Functional Ca_L Channels
Patch-clamp recordings were performed to determine if the appearance of _1C subunits in RR arteries was associated with more functional Ca_L channels. In these studies, freshly isolated VSMCs from RR arteries showed higher levels of voltage-gated barium current (I_Ba) through Ca_L channels than VSMCs of LR arteries (Figures 3A and 3B). In both preparations, I_Ba was sensitive to block by 1 µmol/L nifedipine. Cell capacitance was not significantly different between LR and RR VSMCs, averaging 21.9±0.5 (n=13) and 21.6±0.9 pF (n=8), respectively. Current-voltage (I-V) relationships indicated maximal I_Ba densities of 1.99±0.05 (n=13) and 4.82±0.26 pA/pF (n=8) in VSMCs from LR and RR arteries, respectively (Figure 3C). Thus, a 2.42-fold increase in functional Ca_L channel current was detected in VSMCs exposed to high pressure in the RR circulation.

View larger version (28K): [in this window] [in a new window]   Figure 3. A and B, Recordings of IBa currents generated by 8 mV steps from –70 to +50 mV in VSMCs of LR and RR arteries of 28-day banded rats. IBa was blocked by 1 µmol/L nifedipine. Cell capacitance values were 24.4 (LR) and 16.6 pF (RR). C, Averaged I-V relationships showed higher IBa density in VSMCs of RR compared with LR arteries (n=8 and 13). Subsets of VSMCs were treated with 1 µmol/L nifedipine (n=3 and 5). *Values for LR and RR arteries were significantly different (P<0.05) at the same voltage.

Tension-recording assays also detected more functional Ca_L channels in RR arteries. In these studies, RR arteries developed more Ca²⁺-dependent spontaneous tone than LR arteries, which was reversed by 1 µmol/L nifedipine (Figure 4A). Furthermore, whereas LR arteries showed little responsiveness to increasing concentrations of the Ca_L channel agonist Bay K 8644, RR arteries profoundly contracted, a response associated with an increased availability of Ca_L channels (Figure 4B). Overall, RR arteries developed 2.8-fold more nifedipine-sensitive tone than LR arteries (Figure 4C; n=6). In similar vessels, the maximal responses of LR and RR arteries to Bay K 8644 were 12±6% and 81±13% of the contraction elicited by 80 mmol/L KCl, respectively (Figure 4D; n=8). These data further demonstrate that the upregulation of _1C by high blood pressure in vivo increases the availability of functional Ca_L channels in renal VSMCs.

View larger version (21K): [in this window] [in a new window]   Figure 4. Tension-recording traces in LR and RR arteries from a 28-day banded rat. A, RR artery developed more Ca2+-dependent spontaneous tone, which was reversed by 1 µmol/L nifedipine. B, Only the RR artery showed pronounced contractions to Bay K 8644. C, Nifedipine-sensitive tone was 2.8-fold higher in RR compared with LR arteries (n=6). D, Concentration-response curves in response to Bay K 8644 (n=8). Values represent mean±SEM. *Significant difference (P<0.05) between LR and RR arteries.

Depolarization Upregulates _1C in Isolated Arteries
The literature provides few clues regarding possible signaling pathways that may link high blood pressure to Ca_L channel expression. However, membrane depolarization has long been recognized as a functional alteration in VSMCs exposed to chronic hypertension.^8,12 Thus, we considered the possibility that a reduced membrane potential (E_m) in VSMCs may represent an electrical signal that transduces the stimulus of high blood pressure into Ca_L channel expression. First, we used microelectrode methods to confirm that the VSMCs of RR arteries exposed to high pressure were persistently depolarized, indicating that this electrical signal was available to modulate Ca_L channel expression. Because the E_m levels of renal VSMCs cannot be recorded in vivo, LR and RR arteries were microdissected from 28-day banded rats (n=10), cannulated with glass pipettes, and perfused at average pressures of 73±6 and 182±7 mm Hg, respectively (Figure 5A). The perfusion pressure chosen for each artery reflected the blood pressure level measured below or above the banded site in that particular donor animal. Using this approach, microelectrode impalements revealed that VSMCs in the RR arteries were highly depolarized compared with LR arteries of the same animals (Figure 5B). The E_m values in RR and LR arteries from 10 rats averaged –39.5±0.6 mV and –52.4±0.5 mV, respectively (Figure 5C).

View larger version (18K): [in this window] [in a new window]   Figure 5. A, Isolated LR and RR arteries of 28-day banded rats were perfused at their in vivo blood pressure levels. B, Microelectrode recordings of Em revealed more depolarized VSMCs in RR arteries. C, Em values averaged –52.4±0.5 and –39.5±0.6 mV in LR and RR arteries, respectively (n=10 rats). *Significant difference (P<0.05) between LR and RR values.

Subsequent studies examined if membrane depolarization, as an isolated stimulus, could reproduce the abnormal profile of elevated Ca_L channel expression and function that was established by high blood pressure in RR arteries in vivo. Using a vessel culture approach, renal arteries were microdissected from normal rat kidneys, pooled, and cultured for 2 days in DMEM containing either low K⁺ (4 mmol/L) or high K⁺ (30 mmol/L) concentrations. Assuming a VSMC membrane selectively permeable to K⁺, the high K⁺ solution would be predicted to depolarize the renal VSMCs to –40 mV, although the relatively high Na⁺ permeability of VSMC membranes suggests that this value should be cautiously interpreted.²⁰ Five sets of arteries preconditioned in high K⁺ (Pre-K⁺) demonstrated a 5.47-fold increase in _1C expression compared with control (Ctrl) arteries preconditioned in low K⁺ media for 2 days (Figure 6A). In contrast, _1C was not upregulated in 2 sets of arteries preconditioned in DMEM containing 30 mmol/L sucrose (Figure 6B), indicating that the depolarizing rather than the osmotic challenge of high K⁺ triggered the upregulation of _1C. Notably, Pre-K⁺ VSMCs showed increased levels of nifedipine-sensitive I_Ba through Ca_L channels compared with Ctrl VSMCs (Figure 6C, 6D). Average I-V relationships revealed peak I_Ba densities of 2.66±0.19 (n=7) and 7.41±0.40 pA/pF (n=5) in VSMCs from Ctrl and Pre-K⁺ arteries, respectively, indicating a 2.78-fold increase in I_Ba density (Figure 6E). In the same preparations, average cell capacitance values of 20.2±1.0 and 24.5±0.6 pF were not significantly different.

View larger version (33K): [in this window] [in a new window]   Figure 6. A, 1C (200 to 240 doublet) was upregulated in arteries preconditioned for 2 days in 30 mmol/L K+ (Pre-K+) compared with control arteries (Ctrl) exposed to 4 mmol/L K+. The 45-kDa bands represent -actin. B, Arteries preconditioned for 2 days in high sucrose did not increase 1C expression. C and D, IBa currents generated by 8-mV steps from –70 to +50 mV were higher in Pre-K+ compared with Ctrl VSMCs. IBa was blocked by 1 µmol/L nifedipine. Cell capacitance values were 18.1 (Ctrl) and 22.5 pF (Pre-K+). E, Averaged I-V plots of IBa density for Ctrl and Pre-K+ VSMCs (n=7 and 5), and in subsets of VSMCs treated with nifedipine (n=3 and 4). *Values for Ctrl and pre-K+ VSMCs were significantly different (P<0.05).

Interestingly, arteries preconditioned by depolarization for 2 days developed the contractile phenotype normally reserved for arteries exposed to high blood pressure in vivo.^5,12 Pre-K⁺ arteries demonstrated accentuated levels of nifedipine-sensitive tone in tension-recording studies (Figure 7A), and contracted strongly to Bay K 8644 (Figure 7B), indicating a higher level of functional Ca_L channels. Overall, Pre-K⁺ arteries developed 3.23-fold more nifedipine-sensitive tone than Ctrl arteries (Figure 7C; n=6), and the maximal responses of Ctrl and Pre-K⁺ arteries to Bay K 8644 were 26±13% and 90±9% of the contraction elicited by 80 mmol/L KCl, respectively (Figure 7D; n=5).

View larger version (21K): [in this window] [in a new window]   Figure 7. Tension-recording traces in isolated arteries preconditioned for 2 days in control low K+ (Ctrl) or high K+ (Pre-K+) media. A, Pre-K+ artery developed more spontaneous tone than a Ctrl artery, which was reversed by 1 µmol/L nifedipine. B, Another Pre-K+ artery showed amplified contractions to Bay K 8644. C, Nifedipine-sensitive tone was 3.23-fold higher in Pre-K+ arteries compared with Ctrl arteries (n=6). D, Concentration-response curves to Bay K 8644 in Ctrl and pre-K+ arteries (n=5). All values are mean±SEM. *Significant difference (P<0.05) between Ctrl and Pre-K+ values.

	Discussion

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This study has two principal new findings. First, high blood pressure upregulates functional Ca_L channels in small rat renal arteries by promoting _1C subunit expression. Thus, we have identified a major physiological factor that influences the expression of vascular Ca_L channels in vivo. Second, the depolarization of VSMCs, a fundamental response of the vasculature to high blood pressure, is a powerful stimulus for _1C upregulation. We interpret this finding to suggest that Ca_L channel expression in VSMCs is coupled to the E_m level, and that pressure-induced depolarization of VSMCs is a possible mechanism for the upregulation of _1C subunits during hypertension.

Pressure Dependence of Ca_L Channel Expression
The present study directly demonstrates for the first time that high blood pressure dynamically increases the expression levels of the vascular Ca_L channel _1C subunit in vivo. The key observation that two different levels of _1C expression can be established in the left and right renal circulations of a single animal by exposing them to low and high perfusion pressures, respectively, strongly suggests that blood pressure is a powerful regulator of vascular Ca_L channel expression. Additionally, the VSMCs of RR arteries developed elevated Ca_L channel current and Ca²⁺-dependent tone, implying the appearance of functional Ca_L channels. Interestingly, the _1C short form was particularly sensitive to pressure-induced upregulation at 28 days. This channel form results from a posttranslational proteolytic cleavage of the C-terminus and is missing critical residues required for cAMP-dependent phosphorylation of the Ca_L channel.²¹ Because the latter event mediates vascular relaxation, the induction of the _1C short form by high blood pressure may be relevant to the blunted cAMP-mediated vasodilator responses observed in hypertensive disease.²²

Notably, our finding of a causal, positive relationship between blood pressure and Ca_L channel expression may help to explain why arteries from diverse rat models of hypertension, which rely on different mechanisms to elevate blood pressure, share the common features of increased Ca_L current and Ca²⁺-dependent tone.^6–12 In these animals, high blood pressure may represent a common driving force for vascular _1C expression. In addition, although essential hypertension in humans evolves from diverse mechanisms, an elevated vascular resistance mediated by Ca_L channels is a hallmark finding,²³ and isolated resistance arteries from these individuals show enhanced contractions mediated by Ca_L channels.²⁴ Although rigorous evidence is lacking, these findings also are consistent with an elevated expression of vascular Ca_L channels.

Depolarization as a Signal for Ca_L Channel Expression
Membrane depolarization has long been recognized as a functional characteristic of VSMCs exposed to acute and chronic increases in blood pressure.¹² For example, rat renal arteries depolarize by 15 mV during a pressure elevation from 60 to 140 mm Hg,¹⁵ a response that may be maintained during chronic hypertension.^8,12 Indeed, microelectrode measurements in the present study provide new evidence that depolarization persists in renal VSMCs exposed to high blood pressure in vivo, and therefore, this electrical signal represents a possible event involved in Ca_L channel upregulation. To support this concept, we further demonstrated that simply preconditioning isolated renal arteries in high K⁺ media for 2 days triggered the appearance of _1C protein and functional Ca_L channels. Although a definitive link between blood pressure, VSMC depolarization, and the appearance of vascular Ca_L channels cannot be concluded from these data, the distinctive profile of _1C overexpression, increased Ca_L channel current, and abnormal Ca²⁺-dependent tone induced by depolarization in isolated renal arteries was qualitatively indistinguishable from the profile of Ca²⁺ channel abnormalities induced by high blood pressure in the same arteries in vivo.

In this respect, several earlier reports indicate that abnormal patterns of electrical activity may regulate the expression of Ca_L channels in excitable cells from skeletal and cardiac tissues. For example, Pauwels et al¹⁶ originally demonstrated that exposing chick skeletal myotubes to high K⁺ triggered the appearance of dihydropyridine binding sites within 24 hours. More recently, atrial fibrillation in dogs and humans has been linked to a reduced Ca_L channel current in the affected myocytes,²⁵ an abnormality that may contribute to impaired atrial contraction. However, in the latter condition, and also in other cardiac diseases including left ventricular hypertrophy and failure,²⁶ the molecular basis of altered Ca_L channel availability is a matter of intense debate and only some investigators have detected changes in _1C protein corresponding to altered Ca_L channel availability.^25,26 In contrast, the present study indicates that the expression level of the vascular _1C protein, a splice variant of the cardiac Ca_L channel,³ is highly responsive to the E_m level in isolated small arteries and also is dynamically regulated by blood pressure in vivo. These apparently diverse findings regarding the mechanisms of _1C regulation in vascular and cardiac cells may relate, in part, to the presence of different tissue-specific promoters in cardiac myocytes and VSMCs that drive _1C subunit expression.²⁷

A fundamental question related to the present study is how changes in E_m are transduced into altered Ca_L channel expression. Although a definitive answer will rely on detailed studies in VSMC systems amenable to molecular manipulation, several new findings may provide potential mechanistic insight. Wellman et al²⁸ recently linked E_m to gene transcription in small arteries, by demonstrating that depolarization-induced elevations of intracellular Ca²⁺ ([Ca]_i) activate early response elements and c-fos in VSMCs. In cultured cardiac myocytes, elevated [Ca]_i transiently increases _1C mRNA and upregulates functional Ca_L channels,²⁹ raising the possibility that depolarization-induced increases in [Ca]_i may influence Ca_L channel expression possibly by using a smooth muscle-specific _1C promoter.²⁷ However, posttranslational events including changes in _1C trafficking and stability in the plasma membrane also may influence Ca_L channel availability, and at least two ß subunits exist in VSMCs that may coassemble with _1C to form functional Ca_L channels.^30,31 Regardless, the molecular composition of the Ca_L channel complex in resistance arteries from critical vascular beds has not been delineated, and the physiological factors that regulate _1C expression in the microvasculature remain primarily a mystery.

Study Limitations
Several limitations of the present investigation should be acknowledged. First, patch-clamp experiments were designed to evaluate the level of functional Ca_L channels in VSMCs, and detailed analyses of channel properties and kinetics were not included. These latter factors may have influenced the availability of Ca_L channel current in our experiments, although evidence for Ca_L channel defects in hypertension is lacking.¹² Second, the experiments in the present study in which arteries were exposed to high K⁺ media, simply addressed the question of whether depolarization as an isolated signal could influence vascular _1C expression. These studies clearly could not recapitulate the complex spatiotemporal pattern of signaling events that high blood pressure initiates in small arteries,¹ and other signaling components of the myogenic response should be viewed as potential modulators of Ca_L channel availability. Third, although our data indicated a positive relationship between blood pressure and _1C expression, the possibility exists that other physiological factors influenced _1C upregulation. In this regard, Gerzanich et al³² recently reported that angiotensin (Ang II) infusion in rats enhanced Ca_L channel current in cerebral VSMCs in the absence of _1C upregulation or blood pressure elevation, an event possibly related to mislocalization of endothelial nitric oxide synthase (eNOS). These findings clearly differ from those of the present study in aortic-banded rats, an animal model also characterized by elevated Ang II,¹³ in which LR arteries exposed to high Ang II levels but normal blood pressure showed no evidence of increased Ca_L channel function.

Pressure-Induced Upregulation of Ca_L Channels: A New Paradigm?
The fundamental reaction of small arteries to high blood pressure is the "myogenic response", whereby the VSMCs strongly depolarize and constrict in an effort to normalize blood flow to distal tissues.¹ In the renal circulation, this response will cause further rises in systemic blood pressure, but the enhanced Ca²⁺-dependent vascular tone is relied on at the local level to protect the kidneys from the damage that would occur if the high systemic pressure were transmitted unabated to the glomerular capillary beds.³³ In fact, numerous studies have demonstrated that an increased myogenic tone mediated by Ca_L channels is central to the protection of the kidney from high systemic pressures, and that a loss of this protective vasoconstriction predisposes to progressive glomerular injury.³³ Under these conditions, in which the integrity of the glomerular capillaries depends on a continued strong constriction of the renal circulation, the hypothesis that pressure-induced depolarization upregulates Ca_L channels to facilitate the voltage-dependent influx of activator Ca²⁺ for contraction is appealing from the physiological perspective.

	Acknowledgments

 
Grant support was provided by R01 HL064806 from the National Institutes of Health.

	Footnotes

 
Original received March 2, 2004; revision received April 26, 2004; accepted April 27, 2004.

	References

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