The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1–100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.
- myogenic tone
- Milan normotensive rats
primary (essential) hypertension is a multifactorial disorder that leads to severe cardiovascular and renal complications (39). Hypertension is caused by the complex interplay between genetic predisposition (genetic heritability ∼30%) and multiple environmental factors, including excess dietary salt (1, 46, 70). One major difficulty in identifying genes contributing to hypertension is the etiological heterogeneity of hypertension (1). Polymorphisms in genes encoding proteins involved in the renin-angiotensin-aldosterone system and volume and/or Na+ homeostasis have been the most extensively studied (1). Accumulating evidence indicates that alterations in the genes encoding adducin, a cytoskeleton protein, are associated with enhanced salt retention by the kidneys and hypertension in rats and humans (8, 17, 49, 68).
The Milan hypertensive strain (MHS) of rats is a genetic model of hypertension in which cardiovascular phenotypes apparently depend, at least in part, on adducin gene polymorphisms (11). In the Milan normotensive strain (MNS) × MHS F2 hybrid population, mutation of the Add1 gene accounts for ∼50% of the blood pressure (BP) difference between MHS and its MNS counterpart (10, 11). Adducin polymorphisms have been linked to increased renal Na+ pump activity and enhanced constitutive renal tubular Na+ reabsorption in both rats and humans (23, 27–29, 51, 67). A transient phase of increased salt retention in MHS rats, due to the augmented renal Na+,K+-ATPase activity, is observed in the prehypertensive phase (9). Moreover, after the development of hypertension MHS rats exhibit upregulation of renal apical Na+-Cl− cotransporter and basolateral Cl− channels (15). Thus the MHS rat may be a good model for a salt-dependent form of hypertension. The relationship between the alteration in the adducin genes and the renal dysfunction has been extensively studied (10, 17, 27, 48, 65, 71). The possible vascular functional abnormalities that can contribute to the elevated BP in MHS rats have, however, been largely ignored.
Ca2+ homeostasis plays a crucial role in the genesis of vascular myogenic tone, and increases in arterial smooth muscle cell (ASMC) Ca2+ signaling appear to underlie at least part of the increased peripheral vascular resistance in hypertension (66, 75). Recently we demonstrated that resting cytosolic free Ca2+ concentration ([Ca2+]cyt) and Ca2+ entry via receptor-operated channels (ROCs) and Na+/Ca2+ exchanger-1 (NCX1) are augmented in freshly dissociated mesenteric artery myocytes from MHS rats (79). This is associated with greatly upregulated expression of several cation transport proteins including: C-type transient receptor potential protein (TRPC6; ∼3-fold), an essential component of ROCs [nonselective cation channels that admit Na+ and Ca2+ (56)], NCX1 (∼13-fold), and SERCA2 (∼3-fold) in MHS mesenteric arteries, compared with MNS arteries (79). Ouabain, administered either in vivo or in vitro, also increases expression of these proteins (59). Furthermore, MHS rats have elevated plasma levels of endogenous ouabain (EO) (25, 26), an adrenocortical hormone (34) and a Na+ pump inhibitor. The implication is that the augmented expression of these Na+ and Ca2+ transporters in MHS arteries (79) might be triggered by the high plasma EO. This is important because NCX1 normally mediates Ca2+ entry, rather than exit, in ASMCs of arteries with tone (36). Upregulation of NCX1 should therefore tend to accelerate Ca2+ entry and promote net Ca2+ gain. The increased ROC (TRPC6) and NCX1 expression and Ca2+ signaling in MHS arterial myocytes should enhance myogenic responses and agonist-induced vasoconstriction. This, however, has not been tested.
Here, using the MHS model, we tested the hypothesis that enhanced Na+ reabsorption by the kidneys is linked to 1) altered arterial Ca2+ transport and signaling proteins, 2) augmented small artery tone and evoked constriction, and, thus, 3) BP elevation. The results revealed augmented myogenic reactivity and phenylephrine (PE)-induced vasoconstriction in MHS arteries. This altered arterial function likely contributes to the increased total peripheral vascular resistance (TPR) and elevated BP in MHS rats. Our results contradict the view that, in hypertension, “functional changes in the vasculature have been looked for but have in general not been found, with the exception of a possible endothelial dysfunction” (55).
All experiments were carried out according to the guidelines of and were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
MHS and MNS rats were obtained from Prassis Sigma Tau Research Institute (Milan, Italy); they were bred in the Animal Care Facility of the University of Maryland, School of Medicine. Animals were maintained in a temperature- and humidity-controlled room with a 12-h:12-h light/dark cycle. Rats had free access to tap water and were fed standard rat chow ad libitum. Genomic DNA was obtained from tail biopsies for genotyping by PCR.
We studied male MHS and MNS rats at 10–12 wk of age. Rats were acclimated to the procedures of tail-cuff BP measurements for a week preceding actual data collection. Systolic BPs in MHS and age-matched MNS controls were recorded by tail-cuff plethysmography using a commercial photoelectric system (Model 29 Blood Pressure Meter/Amplifier; IITC, Woodland Hill, CA) that provided constant rates of cuff inflation and deflation as described (79).
Measurements of arterial function.
The rats were euthanized by CO2 overdose followed by decapitation. The mesenteric arcade from a euthanized rat was rapidly dissected and placed in a temperature-controlled dissection chamber (5°C) containing a solution of the following composition (in mM): 145 NaCl, 4.7 KCl, 1.2 MgSO4, 2.0 MOPS, 0.02 EDTA, 1.2 NaH2PO4, 2.0 CaCl2, 5.0 D-glucose, 2.0 sodium pyruvate, and 1% albumin (pH 7.4 at 5°C). Small (4th order) arteries from the superior mesenteric artery arcade were isolated and 3- to 5-mm-long segments without branches were dissected (74). The artery segments were transferred to a recording chamber and were cannulated at both ends (60). One cannula was connected to a servo-controlled pressure-regulated device (Living Systems; Burlington, VT), whereas the other was attached to a closed stopcock. The arteries were pressurized to 70 mmHg (with no internal flow) and were superfused with gassed Krebs solution at 35°-37°C to permit myogenic tone (MT) to develop (60, 74). The Krebs perfusion solution contained (in mM) 112 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11.5 D-glucose, and 10 HEPES (pH 7.4; gas composition: 5% O2-5% CO2-90% N2).
Arteries were viewed through a 10× objective with bright-field illumination on a Nikon TMS microscope (Nikon, Melville, NY) equipped with a monochrome video charge-coupled device camera. The outer diameter was monitored by a real-time edge-detection system that utilizes a video frame grabber and custom-designed LabView software (National Instruments, Austin, TX). The magnitude of the myogenic constriction is expressed as a percentage of the passive external diameter (PD), measured in Ca2+ free Krebs solution at the end of each experiment, i.e., MT = (myogenic constriction/PD) × 100 (74, 75). PE-induced vasoconstriction is expressed as the evoked vasoconstriction relative to PD: Vasoconstriction = [(PD − constricted diameter)/PD] × 100 (74).
Freshly dissociated ASMCs for Ca2+ imaging.
Myocytes were isolated from rat mesenteric arteries (7). The superior mesenteric artery from a euthanized rat was rapidly removed and placed in ice-cold physiological salt solution 1 (PSS1) with the following composition (in mM): 140 NaCl, 5.36 KCl, 0.34 Na2HPO4, 0.44 K2HPO4, 10 HEPES, 1.2 MgCl2, 1.8 CaCl2, and 10 D-glucose (pH 7.2). The mesenteric artery was cleaned of fat and connective tissue and digested in low-Ca2+ (0.05 mM) PSS1 containing (in mg/ml) 2 collagenase type XI, 0.16 elastase type IV, and 2 BSA (fat free) for 35 min at 37°C (7). A suspension of single cells was obtained by gently triturating the tissue in low-Ca2+ PSS1. Dispersed cells were directly deposited on glass coverslips for fluorescence microscopy. Cells were allowed to settle on the coverslips for 20–30 min before loading with fura-2 (79). Freshly dissociated cells that were markedly contracted under resting conditions (less than 5%) were excluded.
[Ca2+]cyt was measured with fura-2 by using digital imaging as described previously (7). Freshly dissociated ASMCs were loaded with fura-2 by incubation for 35 min in PSS1 containing 3.3 μM fura-2 AM (20°-22°C, 5% CO2-95% air). After dye loading, the coverslips were transferred to a tissue chamber mounted on a microscope stage, where cells were superfused for 15–20 min (35° to 36°C) with physiological salt solution 2 (PSS2) to wash away extracellular dye. The PSS2 contained (in mM) 140 NaCl, 5.0 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (pH 7.4). In Ca2+-free PSS2, 50 μM EGTA was added to chelate residual Ca2+. Cells were studied for 40–50 min during continuous superfusion with PSS2 (35°C).
The imaging system included a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY). The dye-loaded cells were illuminated with a diffraction grating-based system (Polychrome V; TILL Photonics, Germany). Fluorescent images were recorded with a CoolSnap HQ2 charge-coupled device camera (Photometrics, Tucson, AZ). Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Molecular Devices, Downingtown, PA). [Ca2+]cyt was calculated by determining the ratio of fura-2 fluorescent emission (510 nm) excited at 380 and 360 nm as described (7). Intracellular fura-2 was calibrated in situ in freshly dissociated ASMCs (7).
Membrane proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) as described (7) and transferred electrophoretically to a nitrocellulose membrane (Amersham BioSciences). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at room temperature with rabbit polyclonal anti-inositol 1,4,5-trisphosphate (IP3) receptor type I antibodies (IP3R-I; dilution 1:750; Affinity BioReagents, Golden, CO). Gel loading was controlled with monoclonal anti-β-actin antibodies (Sigma-Aldrich, St. Louis, MO). After being washed, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h at room temperature. The immune complexes on the membranes were detected by enhanced chemiluminescence-plus (Amersham BioSciences, Piscattaway, NJ) and exposure to X-ray film (Eastman Kodak, Rochester, NY). Quantitative analysis of immunoblots was performed by using a Kodak DC120 digital camera and one-dimensional Image Analysis Software (Eastman Kodak).
Fura-2 AM was obtained from Molecular Probes (Invitrogen Detection Technologies, Eugene, OR). Collagenase (type 2) was obtained from Worthington Biochemical (Freehold, NJ). ATP, PE, ouabain, nifedipine, dimethylsulfoxide, collagenase (type XI), elastase (type IV), and BSA were purchased from Sigma-Aldrich. All other reagents were analytic grade or the highest purity available.
The numerical data presented in RESULTS are means ± SE; n denotes the number of arteries (1 artery per rat) or the number of single cells studied (1 value per cell). The number of animals is presented where appropriate. Data from 12 to 18 rats were obtained for most protocols. Statistical significance was determined using Student's paired or unpaired t-test, or two-way ANOVA, as appropriate. P < 0.05 was considered significant.
Augmented myogenic responses in MHS arteries.
MHS rats have significantly higher mean systolic BP (145 ± 1 mmHg, n = 16) than control MNS rats (112 ± 1 mmHg, n = 16; P < 0.001). To determine the contractile properties of the small resistance arteries from control MNS and MHS rats, the diameter changes in pressurized mesenteric fourth-order arteries (∼165 μm internal diameter) were measured. The arteries constrict spontaneously (MT) when intralumenal pressure is increased to 70 mmHg during superfusion with Krebs solution at 37°C (74, 75). The mean MT in arteries from control MNS rats corresponded to a constriction of 14.9 ± 1.7% of PD (n = 15). Mean MT in arteries from MHS rats was 19.5 ± 1.4% of PD (n = 16), which was significantly greater (P < 0.05) than in controls.
The myogenic responses to step increases in intraluminal pressure [myogenic reactivity (MR)] were augmented in MHS arteries relative to myogenic responses in MNS arteries (Fig. 1). Figure 1A shows a set of original data from a MNS rat artery; the intralumenal pressure was lowered from 70 to 10 mmHg and then raised to 130 mmHg in 20-mmHg increments. Normalization of the myogenic reactivity curves from MHS and MNS arteries to PD at 70 mmHg of the respective arteries [i.e., classic MR curves (69)] reveals that the MHS rat arteries exhibited significantly augmented myogenic constriction (Fig. 1B).
PE-induced Ca2+ responses and vasoconstriction are increased in MHS arteries.
Freshly dissociated mesenteric artery myocytes from the MHS rats had significantly higher resting [Ca2+]cyt than did MNS myocytes (97 ± 2 nM vs. 78 ± 3 nM; P < 0.001) (79). Activation by 5 μM PE, in physiological media, induced augmented Ca2+ signals in the MHS ASMCs. Both the peak initial response, which is likely the result of IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release, and the later plateau, apparently mediated by Ca2+ entry through ROCs and store-operated channels (SOCs), were greater in MHS than in MNS rat ASMCs (Fig. 2, A and B). The plateau was frequently accompanied by low amplitude [Ca2+]cyt oscillations or fluctuations in both MHS and MNS arterial myocytes (Fig. 2A). Superimposed records of the PE-induced Ca2+ response show that the integral of the rise of [Ca2+]cyt (area under the [Ca2+]cyt curve) in MHS arterial myocytes was increased to 142 ± 9% of the area in MNS ASMCs (n = 18 MNS cells and 14 MHS cells; P < 0.05). Similar results were previously obtained with the purinergic receptor agonist, ATP (79).
ASMC contraction depends directly upon the [Ca2+]cyt and on modulation of the sensitivity to Ca2+ (e.g., by Rho/Rho kinase pathway) (30). To determine whether augmented PE-evoked Ca2+ signals are reflected in alteration of PE-induced vasoconstriction of isolated MHS mesenteric small arteries, we compared PE cumulative dose-response curves in pressurized MNS and MHS rat arteries. Figure 3A shows a representative cumulative PE concentration experiment from a control MNS rat artery. The peak vasoconstrictions to 1–100 nM PE were significantly larger in MHS than in MNS arteries (Fig. 3B), but the maximal constriction to 10 μM PE was comparable in MHS and MNS arteries. Thus the apparent EC50 (PE concentration for a half-maximal effect) was significantly lower in MHS than in MNS arteries: 176 ± 39 nM vs. 371 ± 77 nM (P < 0.05).
Augmented SR Ca2+ stores and vasoconstrictor-induced Ca2+ entry in MHS rat arterial smooth muscle.
If agonist-evoked Ca2+ responses and vasoconstriction are increased in MHS rat arterial smooth muscle, it is logical to ask what roles SR Ca2+ release and vasoconstrictor-induced Ca2+ entry play in this augmentation. To address this issue, Ca2+ transients were evoked in freshly dissociated ASMCs by unloading the SR Ca2+ stores in Ca2+-free medium with the vasoactive agonist, ATP, which triggers IP3 synthesis. The peak amplitude of the 5 μM ATP-induced Ca2+ transients in Ca2+-free medium, presumably due to IP3-mediated release from the SR, was significantly larger in MHS than MNS myocytes (Fig. 4, A and B). Subsequent restoration of external Ca2+ evoked a secondary rise in [Ca2+]cyt, apparently associated with Ca2+ entry from the extracellular fluid (ECF) via both SOCs and ROCs. To eliminate the contribution of voltage-gated Ca2+ channels to ATP-induced Ca2+ entry, all solutions in the experiments contained 10 μM nifedipine. This secondary rise in [Ca2+]cyt was also significantly larger in MHS than in MNS myocytes (Fig. 4, A and B). Similar results were obtained for PE: 5 μM PE-induced SR Ca2+ release and subsequent extracellular Ca2+ entry were approximately twofold larger in MHS myocytes (238 ± 28 nM and 91 ± 5 nM, respectively; n = 16) than in MNS myocytes (118 ± 19 nM and 49 ± 6 nM, respectively; n = 20; P < 0.05).
Rat mesenteric arteries express IP3 receptor type 1 (Fig. 4C), but not types 2 or 3 (7). Augmentation of ATP- (Fig. 4, A and B) and PE-mediated Ca2+ release in MHS arterial myocytes in Ca2+-free media correlates with the increased expression of IP3 receptor type 1 (Fig. 4, C and D) and SERCA2 (79). The secondary ATP-evoked rise in [Ca2+]cyt, observed when extracellular Ca2+ was restored (Fig. 4A), correlates with the increased ROC activity (79), a consequence of the approximately threefold upregulated expression of TRPC6 in MHS artery smooth muscle (79).
Increased vasoconstrictor-induced SR Ca2+ release and subsequent Ca2+ entry in MHS arterial myocytes are associated with augmented PE-evoked vasoconstrictions of pressurized mesenteric small arteries under the same conditions. Figure 5A shows the protocol for the PE experiments in a MNS artery. The results are summarized in Fig. 5, B and C. They reveal that both PE-evoked vasoconstrictions, the initial response in Ca2+-free medium, due to SR Ca2+ release, and the second response, due to Ca2+ entry when extracellular Ca2+ was restored, were significantly greater in MHS than in MNS arteries.
Ouabain-induced vasoconstriction is increased in MHS arteries.
The plasma EO level is elevated approximately twofold in MHS (vs. MNS) rats (25, 26), but the EO is washed out when the arteries are isolated. Prolonged treatment of rodents with ouabain also induces an ouabain hypertension (52, 59) that is mediated by high ouabain affinity α2 Na+ pumps (20) [α1 is ouabain resistant in rodents (57)] and NCX (19, 20). Moreover, NCX1 is greatly upregulated in de-endothelialized mesenteric arteries of ouabain hypertension rats (59), as well as in arteries of MHS rats (79). To determine whether circulating EO might directly affect vasoconstrictor responses of MHS mesenteric arteries, we tested the effects of acute application of 100 nM ouabain (derived from plants) on pressurized arteries from MNS and MHS rats. This dose of ouabain rapidly and reversibly inhibits α2 Na+ pumps in rodents (57) but has a negligible effect on rodent low ouabain affinity α1 Na+ pumps (75). Figure 6 shows that the isolated, pressurized small MHS mesenteric arteries exhibited significantly greater ouabain-induced constriction than did MNS arteries. The effects were reversible, and the arteries dilated when the ouabain was washed out (Fig. 6A). These data imply that circulating EO (25, 26) in MHS rats could contribute directly to the elevated MT in small resistance arteries in vivo and, thereby, to the elevated total peripheral resistance and BP.
The present study focuses on arterial function (vascular contractile responses) in MHS rats and complements our earlier report on molecular mechanisms responsible for the augmented Ca2+ signaling in ASMCs from these rats (79). The elevated BP in MHS is associated with adducin gene polymorphisms (11) and is linked to increased renal Na+,K+-ATPase activity, which drives high renal tubular Na+ reabsorption (9, 23, 27, 29, 67), and elevated circulating EO (25, 26). The results described here provide direct evidence that arterial function is altered in MHS rats. This strengthens the view that altered arterial function makes a major contribution to the maintenance of hypertension. It also indicates that the function of small resistance arteries is enhanced in a genetic form of salt-dependent hypertension that is known to exhibit increased proximal tubular Na+ reabsorption. Thus this model appears to be of special relevance to individuals with essential hypertension manifested by augmented proximal tubular sodium reabsorption and salt sensitivity (16).
Enhanced Ca2+ signaling augments myogenic responses and PE-evoked vasoconstriction in MHS arteries.
Altered Ca2+ signaling plays a key role in the vascular dysfunction associated with arterial hypertension (36, 66, 75). Ca2+ signaling activates Ca2+ calmodulin-dependent myosin light chain kinase and triggers vasoconstriction (78). Our findings demonstrate that resting [Ca2+]cyt is elevated (79) and vasoconstrictor (PE, ATP)-induced Ca2+ signals are augmented in freshly dissociated mesenteric artery myocytes from the MHS rats (Figs. 2 and 4, A and B). The enhanced signals are due to both vasoconstrictor-evoked release of SR Ca2+ and Ca2+ entry from the ECF. The augmentation of ATP- and PE-mediated SR Ca2+ release in MHS myocytes can be explained by the increased SR Ca2+ store content resulting from overexpression of SERCA2 (79) and, perhaps, the higher resting [Ca2+]cyt (79) and the upregulation of IP3 receptor type 1 (Fig. 4, C and D), which should enhance mobilization of the larger Ca2+ store.
ROCs and NCX1 mediate the enhanced Ca2+ entry in MHS arteries.
The increase in Ca2+ entry from the ECF in MHS myocytes is associated with ROCs and NCX1. The augmented ATP-induced Ca2+ entry (Fig. 4, A and B) correlates with increased activity of ROCs, measured as Ba2+ influx from the ECF activated by the cell-permeable diacylglycerol analog, 1-oleoyl-2-acethyl-sn-glycerol (OAG) (79). These effects are a consequence of greatly upregulated expression of TRPC6 (79), an essential component of ROCs in various ASMCs (35) including rat mesenteric artery myocytes (59). The upregulation of TRPC6 is specific, since expression of other TRPC proteins is not altered in MHS arteries (79).
Several reports indicate that Ca2+ homeostasis in ASMCs is influenced not only by direct Ca2+ entry through TRPC channels but also by Na+ entry through these nonselective cation channels. The entering Na+ and consequent depolarization should promote Ca2+ entry not only via L-type Ca2+ channels but also through NCX1 (3, 22, 24, 58). We cannot, however, exclude the possibility that other Na+-selective channels (6, 21) might also contribute to the Na+ entry that promotes NCX-mediated Ca2+ entry. In contrast with the heart, where NCX1 primarily mediates Ca2+ extrusion (18), in ASMCs, which are partially depolarized in arteries with tone, NCX1 primarily mediates Ca2+ entry (36, 40, 76). The implication is that the upregulated TRPC6 and NCX1 (79) contribute to the enhanced Ca2+ entry and elevated [Ca2+]cyt (79). This augmentes MR (Fig. 1) and arterial tone (36, 40, 75) and, thus, increases peripheral vascular resistance and BP in MHS rats. It is especially noteworthy that ouabain-induced hypertension (59) and human idiopathic pulmonary hypertension also are associated with a marked upregulation of both NCX1 (77) and TRPC6 (73). In mineralocorticoid-salt hypertension, in which endogenous ouabain has been implicated (34), TRPC6 is also upregulated (5) (NCX1 was not studied). This indicates that the upregulation of these Na+ and Ca2+ transporters is a clue to the origin of the augmented arterial responsiveness in many forms of hypertension (31).
We observed not only augmented myogenic constriction of MHS mesenteric resistance arteries (Fig. 1), but also enhanced contractile responses to 1–100 nM PE (Fig. 3). The augmented PE-evoked vasoconstrictions in the absence and presence of extracellular Ca2+ in MHS arteries (Fig. 5, A–C) are consistent with, respectively, the increased vasoconstrictor-evoked SR Ca2+ release and Ca2+ entry from the ECF (Fig. 4, A and B). Enhanced vascular contractile responses to high dose (>5 μM) norepinephrine were observed previously in mesenteric small resistance arteries from MHS rats (61).
Functional versus structural remodeling of arteries in MHS rats.
Some authors have suggested that vascular structural remodeling, including artery narrowing (53, 55), and increased arterial stiffness (14) are responsible for the increased peripheral vascular resistance in hypertension. The structure of MHS mesenteric small arteries is not altered, however, when compared with MNS arteries (61). In our study, both the passive diameter and the maximal response to PE were identical in MHS and MNS arteries. This also implies that the artery structure and contractile apparatus are normal in the MHS arteries. Thus the enhanced responses of MHS small resistance arteries to stretch and to low-dose PE are more likely associated with augmented Ca2+ signaling or functional remodeling than with structural remodeling of the arteries. Functional changes (augmented MR- and PE-evoked vasoconstriction) in the MHS rat arteries are small (Figs. 1 and 3), even though TRPC6 and NCX1 protein expression (79) and Ca2+ signaling are greatly altered (Figs. 2 and 4, A and B). This can be explained by the fact that the relationship between [Ca2+]cyt and vasoconstriction is nonlinear and that in intact arteries, numerous other factors including endothelial dysfunction, with altered release of endothelium-derived relaxing factors, also influence arterial constriction, peripheral resistance, and BP. For example, endothelium-dependent vasodilation in mesenteric small resistance arteries of MHS is significantly impaired compared with age-matched MNS (61). Nevertheless, from Poiseuille's equation (42) even a modest 5% augmentation of constriction in a 100-μm-diameter artery will increase the resistance to flow by >20%—a large effect when translated into total peripheral resistance and BP.
Vasoconstrictor effects of ouabain.
Nanomolar ouabain exerts its acute vasotonic effects by inhibiting the ASMC high ouabain-affinity α2 Na+ pumps (EC50 ≈ 1 nM) (20, 74, 75) which colocalize with NCX1 and TRPC6 in plasma membrane microdomains adjacent to the underlying junctional SR (37, 38, 63, 79). The ouabain-induced rapid Na+ gradient-driven entry of Ca2+ via nearby NCX1 increases SR Ca2+ stores, augments Ca2+ signaling, and constricts pressurized small arteries (12) (Fig. 6A; also see Refs. 60, 75). Infusion of low-dose ouabain in humans also leads to acute vasoconstriction and increased peripheral vascular resistance (54). Because the plasma EO level is elevated in MHS rats (25, 26), we might expect the circulating EO to enhance, directly, Ca2+ signaling, vascular tone, and agonist-evoked vasoconstriction. It is noteworthy that enhanced spontaneous release of norepinephrine from sympathetic nerve terminals in the arterial wall also contributes significantly to acute ouabain-induced vasoconstriction (60).
These rapid vasoconstrictor effects of low-dose ouabain should not be confused, however, with the chronic effects of ouabain on BP, which depend on the amplification of the ASMC Ca2+ signaling through upregulation of NCX1 and TRPC6 (59). We speculate that this upregulation is mediated by an α2 Na+ pump/protein kinase cascade (43). The upregulation is ligand specific. For example, digoxin, another cardiotonic steroid/Na+ pump inhibitor that also augments ASMC Ca2+ signaling (4), does not upregulate NCX1 and TRPC6 expression (80) and does not raise BP (50). Furthermore, when arteries are removed from ouabain-induced hypertensive rats, the aforementioned changes in protein expression remain and contribute to the ex vivo enhanced Ca2+ signaling (59) and the vascular contractile responses (74).
As described here and in our previous report (79), the same proteins are upregulated in arteries isolated from MHS rats. Crucially, primary cultured arterial myocytes from these arteries do not, however, exhibit upregulation of NCX1 and TRPC6 (79). This is consistent with the view that an in vivo circulating factor (EO), responsible for the protein upregulation, is absent from the culture environment. The implication is that the elevated circulating EO in MHS rats in vivo plays an essential role in the induction and maintenance of the observed functional alterations of the rat arteries, including the augmented TRPC6- and NCX1-mediated Ca2+ signaling and vasoconstriction. In this way, chronic and acute nanomolar ouabain affects arterial smooth muscle function in MHS rats in a coherent, integrated fashion. The net effect is larger constrictions in response to PE (Fig. 3B) and nanomolar ouabain (Fig. 6) in arteries from MHS than from MNS rats. In addition, impaired endothelial vasodilator mechanisms in MHS mesenteric small arteries (61) should contribute further to the amplification of arterial constriction by sympathetic (60) and other vasoactive agents.
A mechanism for whole body autoregulation.
Numerous reports implicate alterations in various Ca2+ homeostasis and signaling proteins in the pathogenesis of hypertension (2, 32, 59, 62, 64, 72). We have identified a critical subset of these proteins that link the enhanced reabsorption of Na+ to the sustained elevation of BP. Our study shows that arterial contractility is enhanced in MHS rats, which carry the hypertensive adducin phenotype and have elevated plasma EO. The augmented myogenic and agonist-evoked vasoconstrictions are consistent with the amplified Ca2+ signaling in ASMCs from MHS rats that results from the upregulation of several Na+ and Ca2+ transport proteins, including NCX1, TRPC6, SERCA2, and IP3R1. This appears to be the first report to tie a genetic defect that enhances renal tubular Na+ reabsorption to: 1) altered expression of arterial Ca2+ transporters and signaling proteins, 2) augmented small artery myogenic and agonist-evoked vasoconstrictor responses, and, thus 3) the elevation of BP.
Nearly a half century ago, Borst and Borst de Geus proposed the concept of whole body autoregulation (13). Guyton and colleagues later showed that plasma volume expansion, as may occur with salt retention, initially elevated BP by increasing cardiac output (CO), but the hypertension was then sustained by high TPR with a normal CO (33). The conversion from high CO to high TPR, termed whole body autoregulation, was attributed to the arterial adjustment needed to prevent tissue overperfusion, but no mechanisms for this phenomenon have been identified. We suggest that the upregulated NCX1, TRPC6 channels, and IP3R1, together with increased sympathetic drive (41, 47), are some of the key mechanisms responsible for the whole body autoregulation and high TPR in hypertension.
The mechanisms that lead to the altered arterial protein expression are still unknown, but the changes in protein expression appear to be initiated by the elevated plasma EO. This implies that Na+ pump-protein kinase cascade signaling (44, 45) is intimately involved in these processes. These results emphasize the importance of vascular molecular and functional abnormalities in the development and maintenance of a genetically induced hypertension that is associated with renal dysfunction in tubular Na+ reabsorption.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: C.I.L., E.K., H.R., and A.Z. performed experiments; C.I.L., E.K., H.R., and V.A.G. analyzed data; C.I.L., E.K., H.R., M.P.B., and V.A.G. interpreted results of experiments; C.I.L., E.K., H.R., and V.A.G. prepared figures; C.I.L., E.K., H.R., A.Z., W.G.W., J.M.H., P.F., M.P.B., and V.A.G. approved final version of manuscript; W.G.W., J.M.H., P.F., M.P.B., and V.A.G. edited and revised manuscript; M.P.B. and V.A.G. conception and design of research; V.A.G. drafted manuscript.
This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grants P01-078870 Project 2 (to V. A. Golovina), Project 1 (to M. P. Blaustein) and Project 3 (to W. G. Wier), R01-045215 (to M. P. Blaustein and J. M. Hamlyn), and R01-HL091969 (to W. G. Wier); by a Japan Heart Foundation/Bayer Yakuhin Research grant (to E. Karashima); and by funds from the University of Maryland School of Medicine.
Present address of Eiji Karashima: Dept. of Cardiology, Shimonsoseki City Central Hospital, Shimonsoseki, Japan.
Present address of Alessandra Zulian: Dept. of Biological Science, Univ. of Padova, Padova, Italy.
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