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(Hypertension. 1995;25:731-734.)
© 1995 American Heart Association, Inc.

Articles

Decreased Dihydropyridine Receptor Number in Hypertensive Rat Vascular Muscle Cells

Kent Hermsmeyer; Anita C. White; David J. Triggle

From the Oregon Regional Primate Research Center, Beaverton.

Correspondence to Dr Kent Hermsmeyer, Oregon Regional Primate Research Center, 505 NW 185th Ave, Beaverton, OR 97006.

	Abstract

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Abstract To further investigate the altered function of Ca²⁺ channels in vascular muscle cells in hypertension, a novel fluorescently labeled dihydropyridine was used with ultrahigh-sensitivity photometry to study dihydropyridine binding sites on the surface membrane of living vascular muscle cells from stroke-prone spontaneously hypertensive rats and their normotensive controls. Fluorescent nitrobenzoxadiazol-6-dihydropyridine in concentrations of 1 to 100 nmol/L bound specifically to vascular muscle cells' Ca²⁺ channels, and was displaced by the unlabeled dihydropyridine analogue or nisoldipine (10 µmol/L). Stroke-prone spontaneously hypertensive rat vascular muscle cells showed significantly decreased binding of nitrobenzoxadiazol-6-dihydropyridine compared with normotensive National Institutes of Health rats. Decreased binding of dihydropyridine by vascular muscle cells from stroke-prone spontaneously hypertensive rats (cells that in other studies show increased Ca²⁺ channel function) indicates a change in channel regulation that is possibly due to a deficiency in the inactivation mechanism, consistent with our earlier electrophysiological studies reporting deficiencies in Ca²⁺-dependent inactivation in genetic hypertension. These data demonstrate decreased numbers of localized sites of dihydropyridine binding on the sarcolemma of living vascular muscle cells, and support the hypothesis that Ca²⁺ channel alterations may significantly contribute to the molecular etiology of genetic hypertension.

Key Words: Calcium channels • rats, inbred SHR • sarcolemma • binding sites • hypertension, genetic

	Introduction

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Many unanswered questions remain about how Ca²⁺ crosses cell membranes, although knowledge about the Ca²⁺ channels that account for the electrically gated part of Ca²⁺ influx has progressed remarkably in the past decade. Combined electrophysiological and optical techniques have revealed that Ca²⁺ channels signal many of the functions in a variety of excitable and secretory cells (reviewed by Tsien et al¹ and Bean² ). The Ca²⁺ channel appears to be an elemental structure consisting of a multipart Ca²⁺ channel protein³ that flickers open and closed within milliseconds to admit trigger amounts of Ca²⁺. The Ca²⁺ channel molecule can be rapidly gated by voltage to increase, by several hundredfold, the rate of Ca²⁺ ion entry,^{1 4} and is the same 1,4-dihydropyridine–sensitive Ca²⁺ channel that is important in vascular muscle cells (VMC) for total peripheral resistance.⁵

For several reasons, it is important to explore the spatial definition of Ca²⁺ channels by directly visualizing dihydropyridine (DHP) binding sites. Examples are such questions as whether there are different populations of Ca²⁺ channels, possible spatial bases for channel interactions, and multiple sites of action of Ca²⁺ channel blockers. The high affinity and selectivity of DHP for L-type Ca²⁺ channels⁶ has been the basis for the characterization and isolation of one of the L-type Ca²⁺ channel proteins.⁷ This study uses a newly synthesized fluorescent DHP, high sensitivity detectors, and digital image acquisition to examine the cellular distribution of DHP binding on the sarcolemma of living cells.

Alterations in Ca²⁺ channels are hypothesized to contribute to hypertension,^{5 8 9} and stroke-prone spontaneously hypertensive rats (SHRSP) show the most extreme example of the high blood pressure exaggeration.¹⁰ To compare Ca²⁺ channel DHP binding in SHRSP with that in VMC from normotensive outbred-panel-of-strains National Institutes of Health rats (N/nih) with high sensitivity and resolution, we used two optical systems. One is a literal photon-counting measurement made with a two-stage microchannel plate camera (Hamamatsu) that is orders of magnitude more sensitive (10^-9 lux) than other cameras. The other system is the Leitz laser scan confocal microscope (CLSM), which was selected for its high resolution in the z axis as well as in the x and y axes. With photon counting, the low background and ultrahigh sensitivity permit detection of DHP binding that tentatively represents only a few Ca²⁺ channels. Images acquired by the photon-counting detector allow femtomolar or even attomolar binding quantification in real time, as well as visualization of composites of Ca²⁺ channels. The z-axis resolution in this whole-field (0.8 NA water immersion) image is calculated to be 12 µm. The CLSM is less sensitive than the photon-counting detector, but offers high z-axis resolution (0.5 µm with the 1.0 NA water immersion objective). The probe used to explore sites of high-affinity binding of DHP is the brightly fluorescent nitrobenzoxadiazol (NBD)-labeled DHP custom synthesized for this purpose. The NBD-6-DHP probe is closely related to nitrendipine. A nonfluorescent analogue of NBD-6-DHP was also available with the same structure but lacking the NBD fluorescent group.

	Methods

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Primary cultures of aortic and azygos vein vascular muscle cells were prepared from neonatal (1 to 4 days old) SHRSP and outbred normotensive control N/nih. Systolic blood pressures of SHRSP averaged 196±11 mm Hg at 8 to 12 weeks, and averages for N/nih were 122±8 mm Hg (n=30 of each animal type). Aortas and azygos veins were dissected from SHRSP and N/nih and placed in 25°C CV3M solution, which consists of 85% minimal essential medium and 15% horse serum (vol/vol), to which is added 4 mmol/L L-glutamine, 20 mmol/L HEPES at pH 7.3, 4 mmol/L NaHCO₃, and 20 µg/mL gentamicin.¹¹ After the rinse in CV3M, vessel segments were soaked for 10 minutes in KG solution, which consists of (mmol/L) potassium glutamate 140, HEPES 25 (final pH 7.3), NaH₂PO₄ 0.5, NaHCO₃ 4, glucose 5.5, and phenol red 0.014. Pieces of each blood vessel type from all the pups of a single litter were minced into 1-mm (maximum dimension) pieces with fine dissecting scissors and incubated at 37°C for 30 minutes in collagenase (30 mg in 10 mL KG solution containing 0.1 µmol/L Ca²⁺). After 30 minutes, the collagenase supernatant was discarded and the sediment was sequentially exposed to three to four incubations of 1 mg/mL trypsin in KG solution for 15 minutes. After each 15-minute incubation in trypsin, the supernatant was removed and placed in 25 mL CV3M on ice. Supernatants were centrifuged at 200g and 4°C for 15 minutes and the pellet was resuspended in 10 mL ice-cold CV3M, which was further centrifuged (always at 4°C) for 10 minutes at 200g. After the supernatant was removed, the remaining cells were resuspended in 10 mL 37°C CV3M, transferred to Falcon plastic T25 tissue culture flasks (Becton Dickinson and Co) for 1 hour of sedimentation, and placed in the incubator at 37°C. After 1 hour, nonattached VMC were transferred to centrifuge tubes, separated at 200g, resuspended in CV3M plus 250 µmol/L bromodeoxyuridine, diluted to a density of about 70 000 cells/mL, and plated on clean 9x22 mm glass coverslips (Corning, Inc) and 35-mm sterile Falcon plastic Petri dishes. VMC were kept in 95% air, 5% CO₂ at 95% relative humidity and 37°C for 2 to 7 days. Bromodeoxyuridine and gentamicin were always removed at least 24 hours before contracting VMC were selected for the binding experiments. Cell culture solutions were from GIBCO, collagenase was from Worthington, and other solutions were from Sigma Chemical Co.

Fluorescence labeling of the VMC was carried out with NBD-6-DHP, the nitrobenzoxadiazol fluorescent derivative of a DHP similar to nitrendipine, but with a CF₃ group replacing the NO₂ group to add photostability (Fig 1). NBD-6-DHP was synthesized by Dr N. Baindur (N. Baindur, unpublished data, 1994). Living, contracting VMC were optimally labeled in room-temperature (22°C) ionic solution for mammals (ISM) (consisting of [mmol/L] NaCl 130, NaHCO₃ 16, NaH₂PO₄ 0.5, KCl 4.7, CaCl₂ 1.8, MgCl₂ 0.4, MgSO₄ 0.4, HEPES 13, and dextrose 5.5, pH 7.3 to 7.4) with a 20-minute exposure to 10 to 100 nmol/L NBD-6-DHP. VMC were loaded by stopping the flow through the 300-µL–capacity laminar flow chamber for 10 minutes, during which 20 µL of 0.1 to 100 nmol/L NBD-6-DHP was placed as a drop over the cells in the field of the microscope (Carl Zeiss, Inc). After 10 minutes of loading with stopped flow, suffusion was restored and 10 minutes allowed for washout of excess fluorescence. For definition of nonspecific binding, cells were exposed to 10 µmol/L unlabeled DHP analogue or 1 to 10 µmol/L nisoldipine after the 15 minutes of preincubation with NBD-6-DHP. For optical quantitation and digital image acquisition, cells were briefly exposed (for 10 seconds or less except as noted) to excitation through fluorescence filters, by use of a Zeiss 25x/0.8 NA water objective, with two layers of Omega 485DF22 (485±11 nm) excitation filters, with emission separated from excitation by a 505-nm DRLP dichroic mirror, and viewed through an Omega 530DF30 (530±15 nm) emission filter. In addition, four separate layers of ultraviolet and infrared blocking filters were included in the light path to protect the cells and probe against both ultraviolet and infrared illumination by a suppression factor of more than 10⁸. Measurements of fluorescence intensity were continued for at least a 10-minute period, with illumination for only 10-second periods, never exceeding six times (except as noted). Masking of the illumination beam limited the excited area to the part of the cell being quantified (typically <1000 µm²). The fluophor was stable for at least 60 seconds of cumulative illumination, during which fading of fluorescence was not detectable. Quantitation was carried out using a Hamamatsu (C1966-20) two-stage microchannel plate camera at its highest sensitivity of 10^-9 lux, with data digitally acquired by a Perceptics Biovision image processor. Averages of 30 frames (1 second) provided the best measure of Ca²⁺ channel binding while not interfering with the dynamics of binding. Longer integrations of up to 30 seconds for higher sensitivity were used only for lowest label concentrations (as noted). Confocal images were analyzed by use of a Leitz CLSM (Leica) with the 488-nm laser and a Leitz 50x/1.00 NA water objective. Quantitation of fluorescence was carried out by calibration with known standards, application of background and field scan corrections, and resulting look-up tables for each cell. The calibration procedure is extensive (K.H. et al, unpublished data, 1994); in brief, it consists of NBD-6-DHP measurements in precision 20-µm path length glass capillary tubing (Vitro Dynamics, Inc) filled with the label in perfusion solution and in a second set in which low concentrations of NDB-6-DHP in alcohol are dried on the glass to correspond with sarcolemma labeling. Surface layer fluorescence was expressed as number of moles per square micron (the latter being the approximate area of a fluorescent cluster) and as the absolute amount of fluorphor, stated in moles. Conversion to B_max values was based on Scatchard curve intercepts and expressed as moles of binding site per entire cell. Statistical comparisons were made using ANOVA with Scheffé tests, selecting P<.05 as the criterion for significance.

View larger version (18K): [in this window] [in a new window]   Figure 1. Top, diagram shows structure of the fluorescent label 7-nitrobenz-2-oxa-1,3-diazol-4-yl-6-dihydropyridine (DHP-6-NBD). Bottom, spectra for excitation and emission.

	Results

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Fig 2 shows images of VMC at saturation labeling at 22°C and 30 minutes for SHRSP and N/nih. Fig 2A shows an N/nih aortic VMC that is more strongly labeled than that from SHRSP (Fig 2B), and Fig 2C is an N/nih azygos vein VMC that has more NDB-6-DHP binding than that from SHRSP (Fig 2D) after exposure to 10 nmol/L fluorescent label. These four VMC images are representative of N/nih labeling compared with SHRSP labeling in 9 to 12 studies with each cell type. There were fewer DHP receptors in both the aorta and the azygos vein VMC of SHRSP, as shown in Fig 3, which is a plot of specific binding isotherms for an example of each blood vessel type. Half-saturation was reached by approximately 4 nmol/L of free DHP for all four cell types. Specific binding in N/nih was 81±2% for aorta and 89±4% in azygos vein VMC, and in SHRSP it was 78±5% in aorta and 90±4% in azygos vein VMC (n=8 for each of the four types of VMC). Saturation occurred at about 40 nmol/L for each type, but with a different maximum, which was generally greater than the complete curves in single VMC shown in Fig 3. In Fig 3, only one cell for each of the four types is shown. Scatchard fits of the fluorescence binding data showed differences in the number of DHP receptors, but no difference in affinity (Table). Data in the Table represent composite averages by cell type for all cells studied. B_max was decreased by 50% in aorta and 43% in azygos vein VMC of SHRSP (both significantly different). Ca²⁺ channel labeling was nonuniform at all concentrations in both whole-field and confocal images, suggesting that clustering of Ca²⁺ channels is the predominant pattern. In contrast, nonspecific fluorescence showed a random distribution in each of 32 cells.

View larger version (65K): [in this window] [in a new window]   Figure 2. Photographs of whole-field fluorescence at NA=0.8 show that binding of fluorescent dihydropyridine to vascular muscle cells was greater in normotensive National Institutes of Health rats (N/nih) than in stroke-prone spontaneously hypertensive rats (SHRSP) for both aorta and azygos vein. A, N/nih aorta; B, SHRSP aorta; C, N/nih azygos vein; and D, SHRSP azygos vein. Vascular muscle cells contained clusters of dihydropyridine fluorescence, which were even more evident in confocal images (not shown), nearly all of which (>90%) could be displaced by 10 µmol/L nisoldipine. Both blood vessels were less labeled with the dihydropyridine probe in SHRSP than in N/nih. These data are representative of 9 to 12 experiments for each of four cell types.

View larger version (20K): [in this window] [in a new window]   Figure 3. Line graph shows that stroke-prone spontaneously hypertensive rat (SP-SHR) saturation isotherms at 22°C were depressed compared with normotensive National Institutes of Health rat (N/nih) saturation isotherms for both aorta and azygos vein vascular muscle cells. The plots shown are from one cell for each curve, and at 1, 2, and 5 nmol/L were integrated for 30 seconds (with fluorescence fading). Although these curves show a smaller decrease than the averages from eight cells presented in the Table, the direction of the difference was consistent. Binding sites are expressed as moles per cell. Conversion depended on fluorescence standards prepared as ultrathin layers on glass, with number of moles rather than moles per milliliter as the resulting unit. AFU indicates arbitrary fluorescence units integrated over one cell.

View this table: [in this window] [in a new window]   Table 1. Dissociation Constants and Numbers of Receptors

Ca²⁺ channel clusters were observed at certain points on the sarcolemma, and were clearly not internal or vesicular. Intense fluorescent labeling of areas of various sizes were found over the VMC surface in these whole-field (nonconfocal) images. DHP binding clusters were localized to peripheral (sarcolemmal) sites, as shown by examination with confocal laser scanning. No internalization of fluorescence was detected during the 30-minute observation period, suggesting that all label was at the surface membrane. Specificity of the labeling was additionally implied by displacement to less than 10% of the maximum in 10 minutes on exposure to either nonfluorescent DHP or nisoldipine at 10 µmol/L.

The Table summarizes binding differences of SHRSP compared with N/nih VMC. Although binding occurs with the same affinity in SHRSP and N/nih VMC, there are significantly fewer NBD-6-DHP binding sites in both aorta and azygos vein VMC of SHRSP compared with N/nih. The data suggest that the number of DHP binding sites per VMC may be on the order of several thousand, with a lower number of Ca²⁺ channels associated with VMC of genetically hypertensive rats. The difference is significant at P<.05.

	Discussion

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These experiments demonstrate differences in specific binding of the novel fluorescent probe NBD-6-DHP to the surface membrane of intact, contracting VMC. Decreased binding of DHP to SHRSP VMC compared with N/nih VMC suggests a deficiency in function of Ca²⁺ channels, probably at the inactivation gate, where Ca²⁺ antagonists are believed to bind to the Ca²⁺ channel.³ DHP affinity for VMC Ca²⁺ channels in these normally polarized cells was lower than under depolarized conditions, such as in broken cell preparations.^{6 7} Membrane potential has been measured as -45 to -50 mV in isolated, cultured rat aortic and azygos vein VMC,¹¹ and could account for a 5- to 10-fold lower binding affinity. Fluorescent DHP label was displaced by nisoldipine at 1 to 10 µmol/L, suggesting specific competition for functional DHP binding sites. Nisoldipine was more effective in displacing fluorescent DHP than was the unlabeled analogue, probably reflecting a higher affinity of nisoldipine for these Ca²⁺ channel binding sites (N. Baindur et al, unpublished data, 1994).

SHRSP show exaggerated blood pressures and correspondingly exaggerated Ca²⁺ currents compared with either their N/nih controls or the less hypertensive SHR from which their strain was derived.¹⁰ Although our hypothesis was that Ca²⁺ channel number in SHRSP would be increased, we found the opposite to be the case. Fundamental alterations in the Ca²⁺ channel governing inactivation may be responsible. Studies of Ca²⁺ channel function have shown electrophysiological manifestations of a compromised inactivation process.^{9 10 12 13} Furthermore, evidence from studies of Ca²⁺ agonists that contraction on exposure to the agonist Bay k8644 is stronger in hypertension would also be consistent with deficient inactivation.^{6 7} Perhaps Ca²⁺ channels with slowed inactivation might downregulate through a feedback control process not yet recognized.¹⁴ These data thus suggest a second hypothesis, that Ca²⁺ channel inactivation (perhaps at Ca²⁺ antagonist binding sites) may be diminished in genetic hypertension, at least in these rat genetic models. If this is the case, the notable efficacy of Ca²⁺ antagonists in reducing blood pressure in SHRSP and SHR, despite decreased binding, suggests the fundamental importance of even partial inhibition of Ca²⁺ influx in VMC.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-38537 and by Miles Laboratories, New Haven, Conn.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Tsien RW, Ellinor PT, Horne WA. Molecular diversity of voltage-dependent Ca²⁺ channels. Trends Physiol Sci. 1991;12:349-354.

2. Bean BP. Classes of calcium channels in vertebrate cells. Annu Rev Physiol. 1989;51:367-384. [Medline] [Order article via Infotrieve]

3. Hofmann F, Biel M, Flockerzi V. Molecular basis for Ca²⁺ channel diversity. Annu Rev Neurosci. 1994;17:399-418. [Medline] [Order article via Infotrieve]

4. Catterall WA. Structure and function of voltage-gated ion channels. Trends Neurosci. 1993;16:500-506. [Medline] [Order article via Infotrieve]

5. Rusch NJ, Hermsmeyer K. Vascular muscle Ca²⁺ channels in hypertension. In: Coca A, Garay RP, eds. Ion Transport in Hypertension: New Perspectives. Boca Raton, Fla: CRC Press; 1994;197-227.

6. Triggle DJ. Calcium channel drugs: structure-function relations and selectivity of action. J Cardiovasc Pharmacol. 1991;18(suppl 10):S1-S6.

7. Triggle DJ. Calcium, calcium channels, and calcium antagonists. Drugs Dev. 1993;2:3-13.

8. Hermsmeyer K. Differences of calcium channels in vascular muscle in hypertension. Am J Hypertens. 1991;4:412S-415S. [Medline] [Order article via Infotrieve]

9. Hermsmeyer K, Bian K. Calcium channel hypothesis in hypertension. J Vasc Med Biol. 1991;3:219-222.

10. Self DA, Bian K, Mishra SK, Hermsmeyer K. Stroke-prone SHR vascular muscle Ca²⁺ current amplitudes closely correlate with lethal increases in blood pressure. J Vasc Res. 1994;31:359-366. [Medline] [Order article via Infotrieve]

11. Hermsmeyer K, Mason R. Norepinephrine sensitivity and desensitization of cultured single vascular muscle cells. Circ Res. 1982;50:627-632. [Abstract/Free Full Text]

12. Rusch NJ, Hermsmeyer K. Calcium currents are altered in the vascular muscle cell membranes of spontaneously hypertensive rats. Circ Res. 1988;63:997-1002. [Abstract/Free Full Text]

13. Hermsmeyer K, Rusch NJ. Calcium channel alteration in genetic hypertension. Hypertension. 1989;14:453-456. [Abstract/Free Full Text]

14. Ferrante J, Triggle DJ. Drug and disease induced regulation of voltage-dependent calcium channels. Pharmacol Rev. 1990;42:29-44.[Medline] [Order article via Infotrieve]

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