Decreased Dihydropyridine Receptor Number in Hypertensive Rat Vascular Muscle Cells

Methods

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 9×22 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 25×/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 50×/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.

• Download figure
• Open in new tab
• Download powerpoint

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

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.

• Download figure
• Open in new tab
• Download powerpoint

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.

• Download figure
• Open in new tab
• Download powerpoint

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.

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

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.