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(Hypertension. 1995;25:752-757.)
© 1995 American Heart Association, Inc.

Articles

Calcium- and Protein Kinase C–Dependent Basal Tone in the Aorta of Hypertensive Rats

Michael L. Pucci; Xianglan Tong; Kathleen B. Miller; Hui Guan; Alberto Nasjletti

From the Department of Pharmacology, New York Medical College, Valhalla.

	Abstract

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Abstract We examined the regulatory influence of nitric oxide on development of calcium- and protein kinase C–dependent basal tone in rings of thoracic aortas from rats with aortic coarctation–induced hypertension and from normotensive controls. Aortic rings from hypertensive rats but not those from normotensive rats, bathed in Krebs' bicarbonate buffer and subjected to 2 g of passive stretch, were relaxed by removal of calcium from the buffer and by the protein kinase C inhibitors staurosporine and calphostin C. Protein kinase C activity was much greater in homogenates of aortae from hypertensive rats than in those from normotensive controls (2124±785 versus 608±73 pmol · min^-1 · mg protein^-1, respectively). Relaxant responses to removal of calcium and to staurosporine were greater in aortic rings rubbed to remove the vascular endothelium than in endothelium-intact rings (-1.07±0.12 versus -0.70±0.10 g tension/mg tissue, respectively, for calcium removal and -1.10±0.12 versus -0.65±0.08 g tension/mg tissue, respectively, for staurosporine). Treatment with an inhibitor of nitric oxide synthesis increased calcium-dependent tone in both intact and endothelium-denuded aortic rings from hypertensive rats. Conversely, the administration of sodium nitroprusside or L-arginine reversed tone in both intact and denuded aortic rings from hypertensive rats, but acetylcholine reversed tone only in intact rings. The relaxant effects of these agents were paralleled by increases in cyclic guanosine monophosphate in aortic tissue. We conclude that aortic rings from rats with aortic coarctation–induced hypertension display calcium-dependent, protein kinase C–mediated tone in the absence of exogenous vasoconstrictors. Furthermore, endogenous nitric oxide from endothelial and nonendothelial sources suppresses the development of this tone.

Key Words: hypertension, arterial • muscle, smooth, vascular • nitric oxide • protein kinase C • calcium • vasoconstriction

	Introduction

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Previous studies demonstrated that exposure to calcium-free media elicits relaxation of segments of aortae from spontaneously hypertensive rats (SHR) but not from normotensive rats.^{1 2 3} Recently, we reported that an inhibitor of protein kinase C (PKC) induces relaxation in rings of thoracic aortae from rats with aortic coarctation–induced hypertension of 7 to 14 days' duration, but not in rings from normotensive controls.⁴ These observations indicate that aortic smooth muscle from hypertensive rats displays a high basal tone relative to that from normotensive rats, a tone that is calcium-dependent in SHR and may be PKC-dependent in rats with aortic coarctation.

Inhibitors of nitric oxide (NO) synthesis were reported to promote tension development in aortic smooth muscle from SHR⁵ and rats with aortic coarctation–induced hypertension.⁴ The aortic constrictor responses to inhibitors of NO synthesis in these hypertensive rats may result from a heightening of the mechanisms responsible for implementation of the high basal tone. In this respect, NO is known to serve as a counterregulatory influence to mechanisms of vascular contraction^{6 7} by means of cyclic guanosine monophosphate (cGMP)–mediated mechanisms that reduce intracellular calcium or interfere with its increase during hormonal stimulation.⁸

The present study was undertaken to investigate the regulatory influence of NO on the basal tone of rings of thoracic aortae taken from sham-operated normotensive rats and rats with aortic coarctation–induced hypertension of 7 to 14 days' duration. First, we examined the effects of endothelium removal and an inhibitor of NO synthesis on the expression of calcium- and PKC-dependent basal tone in aortic rings from normotensive and hypertensive rats. Second, we compared aortic rings from normotensive and hypertensive rats, with and without endothelium, in terms of the effects of sodium nitroprusside (SNP), acetylcholine, and L-arginine on basal tone.

	Methods

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Drugs and Solutions
Unless otherwise noted, all drugs and chemicals were from Sigma Chemical Co. Pyrogen-free deionized water was used in the preparation of all buffers and solutions. Stock solutions of acetylcholine, SNP, L-arginine, D-arginine, L-lysine, methylene blue (MB), and N^G-nitro-L-arginine methyl ester (L-NAME) were prepared in deionized water, stored as aliquots at -20°C, and further diluted with Krebs' bicarbonate buffer at the time of the experiments. Concentrated stock solutions of staurosporine, calphostin C (Kamiya Biomedical Co), 3-isobutyl-1-methyl-xanthine (IBMX), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were prepared in dimethyl sulfoxide. The composition of Krebs' bicarbonate buffer (mmol/L) was NaCl 118.5, KCl 4.7, CaCl₂ 2.8, KH₂PO₄ 1.2, MgSO₄ 1.1, NaHCO₃ 25.0, and dextrose 11.1. In some experiments, calcium-free Krebs' bicarbonate buffer was used. The composition was as above except CaCl₂ was omitted and 2 mmol/L EGTA was added.

Animals
Experiments were conducted on male Sprague-Dawley rats (300 to 350 g, Charles River) with aortic coarctation–induced hypertension of 7 to 14 days' duration and on sham-operated, normotensive controls (n=30 for both groups). The rats were prepared according to surgical procedures described in detail elsewhere⁴ and approved by the Institutional Animal Care and Use Committee of New York Medical College.

Measurement of Isometric Tension
On the day of the experiment, rats were anesthetized with sodium pentobarbital (60 mg/kg IP), the carotid artery was cannulated with polyethylene tubing (PE 50), and arterial blood pressure was measured. Mean arterial pressure was elevated in rats 7 to 14 days after aortic coarctation (168±6 versus 105±5 mm Hg for controls, P<.05). After blood pressure measurement, the descending thoracic aortas were removed and prepared as ring segments according to previously published methods.⁴ In some experiments, the lumenal surfaces of aortic rings were rubbed to remove the endothelium. Each aortic ring was placed in a tissue bath filled with Krebs' bicarbonate buffer (37°C), bubbled with 95% O₂/5% CO₂, stretched to 2.0 g of isometric tension, and allowed to equilibrate for 60 to 90 minutes with changes in buffer at 15-minute intervals. In preliminary experiments, we found that 2.0 g of tension was optimal for expression of contractile responses induced by KCl (120 mmol/L) in aortic rings from both groups. In some experiments, L-NAME (3x10^-4 mol/L) was added to the tissue bath from the outset of the equilibration period to inhibit NO synthesis.⁹ When isometric tension was stable, the experiments were performed.

In one series of experiments, we assessed the effects of calcium-free Krebs' buffer on the basal tone of unrubbed and rubbed aortic rings from normotensive and hypertensive rats, with and without L-NAME added to the tissue bath at the onset of equilibration. In a second series of experiments, we examined the effects of staurosporine (10^-8 mol/L) or calphostin C (10^-6 mol/L), potent inhibitors of PKC,^{10 11} on basal tone of unrubbed and rubbed aortic rings from normotensive controls and hypertensive rats. In aortic rings from normotensive rats, these concentrations of staurosporine and calphostin C were sufficient to block contractile responses to a maximally effective concentration of TPA (10^-6 mol/L). In a third series of experiments, we compared the effects of cumulative addition of SNP (an NO-generating agent¹² ), acetylcholine (which stimulates NO synthesis in endothelial cells¹³ ), or L-arginine (the substrate for NO synthesis¹⁴ ) on basal tone of unrubbed and rubbed aortic rings from these normotensive and hypertensive rats. The effects of D-arginine, the inactive enantiomer of L-arginine,¹⁴ or L-lysine, a basic amino acid subject to transcellular transport mechanisms similar to those of L-arginine,¹⁵ on basal tone of unrubbed and rubbed aortic rings from hypertensive rats were assessed in similar experiments. At the end of experiments examining the effects of L-arginine, D-arginine, and L-lysine, SNP (5x10^-6 mol/L) was administered to determine the maximal SNP-inhibitable tone. After the experiments were completed, aortic rings were allowed to dry for at least 24 hours and then weighed. Results are expressed as the change in isometric tension induced by calcium-free buffer or experimental drugs, normalized by the dry weight of the vascular rings.

Measurement of cGMP Levels in Aortic Rings From Normotensive and Hypertensive Rats
Complementary experiments were performed to compare the cGMP content of aortic rings from hypertensive and normotensive rats. In brief, aortic rings (two per condition) were equilibrated in Krebs' bicarbonate buffer containing IBMX (0.5 mmol/L) for 1 hour in a shaking water bath at 37°C. At the midpoint of the equilibration period, MB (10^-5 mol/L), an inhibitor of soluble guanylate cyclase,¹² was added to the appropriate tubes. After equilibration, the buffer was replaced with fresh buffer containing IBMX and the following agents: vehicle (no additions), acetylcholine (10^-6 mol/L) alone, acetylcholine (10^-6 mol/L) and MB (10^-5 mol/L), L-arginine (10^-3 mol/L) alone, L-arginine (10^-3 mol/L) and MB (10^-5 mol/L), and SNP (5x10^-6 mol/L). The rings were then incubated for 5 minutes, after which time they were snap-frozen in liquid nitrogen and stored at -70°C until assay for cGMP according to published methods.^{4 16}

Measurement of PKC Activity in Homogenates of Aortas From Normotensive and Hypertensive Rats
We measured PKC activity in homogenates of aortic tissue from sham-operated normotensive rats and hypertensive rats 7 to 14 days after aortic coarctation according to published methods,¹⁷ with minor modifications. The descending thoracic aortae of rats were excised, cleared of excess blood and periadventitial tissue in ice-cold NaCl solution (0.15 mol/L), snap-frozen in liquid nitrogen, and stored at -70°C until assayed. At the time of assay, aortas (one per assay) were homogenized (Polytron, Brinkman) in TRIS buffer (20 mmol/L, pH 7.5) with EDTA and EGTA (0.5 mmol/L each); leupeptin and aprotinin (0.025 mg/ml each); ß-mercaptoethanol (10 mmol/L); and Triton X-100 (0.5%). Homogenization and all subsequent preparatory steps were carried out at 4°C. Homogenates were incubated for 30 minutes and centrifuged at 10 000g for 15 minutes. Supernatants were then subjected to anion exchange chromatography (DEAE-Sephacel) to partially purify the samples, and column eluates were assayed for total PKC activity with a commercially available kit (GIBCO BRL). In brief, a specific substrate peptide, N-acetylated myelin basic protein fragment 4-14 (0.05 mmol/L), was combined with column eluate (0.5 to 1.5 µg protein) in TRIS buffer (20 mmol/L, pH 7.5) containing MgCl₂ (20 mmol/L), CaCl₂ (1 mmol/L), adenosine triphosphate (ATP) (0.02 mmol/L), and -[³²P]ATP (0.625 µCi per assay tube; specific activity, 3000 Ci/mmol; Du Pont NEN) in the presence of lipid reagent (0.01 mmol/L phorbol 12-myristate-13-acetate, 0.28 mg/mL phosphatidyl serine, and Triton X-100 mixed micelles) or of pseudosubstrate peptide PKC(19-36) and incubated for 5 minutes at 30°C. Phosphorylation of the substrate peptide was assessed by scintillation spectrophotometry of aliquots of incubate that had been placed on phosphocellulose discs and washed with phosphoric acid and water. PKC specific activity was defined as the difference between activity in the presence of lipid reagent versus that in the presence of pseudosubstrate peptide, and was expressed as picomoles of incorporated phosphate per minute per milligram protein (Biorad).

Statistical Analysis
Results are expressed as mean±SEM. Unless otherwise specified, n in figures, legends, and text denotes the number of vascular rings taken from four or more rats. Data were analyzed by paired or unpaired Student's t test or by ANOVA, as appropriate (see figure legends for specific test used). If differences were noted by ANOVA, the Newman-Keuls modified t test was used to make specific comparisons. PKC activity data were subjected to logarithmic transformation before application of Student's t test. The null hypothesis was rejected when the P value was <.05.

	Results

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Fig 1 illustrates the effect of changing from calcium-containing buffer to calcium-free buffer on basal tone of aortic rings from normotensive and hypertensive rats. Calcium-free buffer had no effect on tension of unrubbed or rubbed aortic rings from sham-operated, normotensive rats, but it induced profound relaxation of aortic rings taken from hypertensive rats 7 to 14 days after aortic coarctation. Relaxant responses to calcium-free buffer were greater in rubbed than in unrubbed aortic rings from these hypertensive rats. Also, relaxant responses to calcium-free buffer were increased in both unrubbed and rubbed aortic rings from hypertensive rats that had been pretreated with L-NAME during the equilibration period.

View larger version (21K): [in this window] [in a new window]   Figure 1. Bar graph shows change in basal tone induced by calcium-free Krebs' bicarbonate buffer in unrubbed and rubbed aortic rings from normotensive (open bars) and hypertensive rats (closed bars), after equilibration in the absence (vehicle) or presence of NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 mol/L) for 60 to 90 minutes. Values are mean±SEM. Numbers in parentheses denote the number of vascular rings from four or more rats. *P<.05 relative to unrubbed rings from the same group by paired t test; P<.05 relative to appropriate vehicle control by paired t test.

Staurosporine (10^-8 mol/L) and calphostin C also induced relaxant responses in unrubbed and rubbed aortic rings from hypertensive rats (Fig 2), the relaxations being greater in rubbed than in unrubbed rings. These agents had no effect on tension of rings from normotensive controls. The magnitudes of the responses to staurosporine and calphostin C were virtually identical to those seen with calcium-free buffer. Of note, total PKC activity was increased approximately 3.5-fold in aortic homogenates from hypertensive rats compared with those from normotensive controls (2124±785 versus 608±73 pmol · min^-1 · mg protein^-1; n=9 and n=5, respectively; P<.05).

View larger version (23K): [in this window] [in a new window]   Figure 2. Bar graphs show change in basal tone induced by staurosporine (10-8 mol/L) and calphostin C (10-6 mol/L) in unrubbed (open and shaded bars) and rubbed (closed and hatched bars) aortic rings from normotensive (shaded and hatched bars) and hypertensive (open and closed bars) rats. Values are expressed as mean±SEM. Numbers in parentheses denote the number of vascular rings from four or more rats. *P<.05 relative to unrubbed rings from the same group by paired t test.

Fig 3 shows the effects of SNP, acetylcholine, and L-arginine on basal tone of aortic rings from normotensive and hypertensive rats. No effect on tension of aortic rings from normotensive, sham-operated rats was elicited by SNP (up to 10^-5 mol/L), acetylcholine, or L-arginine. SNP caused relaxation of both unrubbed and rubbed aortic rings from hypertensive rats. At the highest concentrations of SNP, the relaxant response was greater in rubbed than in unrubbed rings. Acetylcholine caused relaxation of unrubbed aortic rings from hypertensive rats but had no effect on tension of rubbed rings. L-Arginine caused relaxation of both unrubbed and rubbed rings from hypertensive rats. Although responses to L-arginine were similar in unrubbed and rubbed rings when expressed as grams of tension per milligram of tissue, the maximal effect of L-arginine was greater in unrubbed rings than in rubbed rings when expressed as a percentage of SNP-inhibitable tone (61±14% versus 28±8%; n=7 and n=5, respectively; P<.05). Neither D-arginine nor L-lysine (10^-5, 10^-4, and 10^-3 mol/L) had relaxant effects on unrubbed aortic rings from hypertensive rats 7 to 14 days after aortic coarctation (-0.01±0.03, -0.05±0.04, and -0.05±0.06 g tension/mg tissue for D-arginine [n=7] and -0.04±0.03, -0.02±0.06, and 0.22±0.14 g tension/mg tissue for L-lysine [n=6], respectively). SNP-inhibitable tone in aortic rings exposed to D-arginine and L-lysine was 0.77±0.23 and 0.68±0.14 g tension/mg tissue, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 3. Bar graphs show change in basal tone induced by sodium nitroprusside (top), acetylcholine (middle), and L-arginine (bottom) in unrubbed (open and shaded bars) and rubbed aortic rings (closed and hatched bars) from hypertensive (open and closed bars) and normotensive rats (shaded and hatched bars). Values are expressed as mean±SEM. For each pharmacological agent, experiments were performed in at least six vascular rings from six different hypertensive rats and in at least four vascular rings from four different sham-operated rats. Statistical analysis was by ANOVA followed by Newman-Keuls modified t test. *P<.05 relative to unrubbed rings from the same group.

Because the vasodilatory effects of NO are mediated by increases of cGMP in vascular smooth muscle,¹¹ we measured cGMP content of aortic rings from normotensive and hypertensive rats in the absence and presence of acetylcholine, L-arginine, or SNP. The Table illustrates cGMP levels in unrubbed aortic rings from these rats. Although basal cGMP levels and the stimulatory effects of acetylcholine and SNP on cGMP were similar in both groups, L-arginine increased cGMP in aortic rings from hypertensive rats 7 to 14 days after aortic coarctation but not in those from normotensive controls. Prior exposure to MB blocked the increases of cGMP caused by acetylcholine in both groups and by L-arginine in aortic rings from hypertensive rats. Of further interest, L-arginine (10^-3 mol/L) also induced a small but significant increase of cGMP in rubbed aortic rings from hypertensive rats (basal level, 3.5±0.6 pmol/mg protein; with L-arginine, 7.7±1.4 pmol/mg protein [n=5; P<.05]). In rubbed aortic rings from both groups, acetylcholine had no effect on cGMP levels, and SNP induced similar increases in cGMP in aortic rings from hypertensive and normotensive rats (244±59 versus 193±50 pmol/mg protein; n=10 and n=9, respectively).

View this table: [in this window] [in a new window]   Table 1. Cyclic Guanosine Monophosphate Levels in Unrubbed Aortic Rings From Normotensive and Hypertensive Rats

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, removal of calcium induced profound reduction of basal tone of aortic rings from rats with aortic coarctation–induced hypertension but not of those from normotensive controls. This indicates that aortic rings from hypertensive rats display a high level of active tone in the absence of exogenous vasoconstrictors and, furthermore, that the tone is calcium dependent. On the other hand, our findings demonstrate that aortic rings from normotensive rats do not display active tone in this setting. Our observations in rats with aortic coarctation–induced hypertension and in normotensive controls parallel previous reports that aortic tissue from SHR but not from Wistar-Kyoto (WKY) rats exhibits calcium-dependent tone in the absence of exogenous stimulatory agents.^{1 2 3} There is abundant evidence that calcium metabolism and vascular smooth muscle reactivity to extracellular calcium and to calcium-elevating vasoconstrictor agents are altered in vascular tissue from hypertensive animals.¹⁸ In aortic rings from rats with aortic coarctation–induced hypertension, both the contractile response¹⁹ and the rise in intracellular free calcium ion concentration ([Ca²⁺]_i)²⁰ induced by -adrenoceptor agonists are accentuated compared with normotensive controls. Also, higher basal [Ca²⁺]_i has been observed in unstimulated vascular smooth muscle cells of SHR²¹ and rats with aortic coarctation–induced hypertension.²² Aberrant calcium metabolism in vascular smooth muscle of hypertensive rats may be linked to altered phospholipase C–mediated inositol phosphate production,²³ altered calcium channel activity,²⁴ altered release of calcium from intracellular stores,²⁵ or altered removal of calcium from the cytosol.²⁶ The mechanisms responsible for the development of calcium-dependent tone in the aortas of hypertensive rats are not clear at this point.

The high resting tone in aortic rings from hypertensive rats 7 to 14 days after aortic coarctation may be linked to activation of PKC. That staurosporine and calphostin C caused relaxation of basally equilibrated aortic rings from hypertensive rats implies that development of active tone in these aortic rings is dependent on a mechanism of vascular contraction mediated by PKC. In support of this notion, we found that total PKC activity in aortic tissue from hypertensive rats was increased more than threefold over that in tissue from normotensive controls. Functional studies in isolated tissues strongly suggest that PKC-mediated mechanisms of vascular contraction are altered in hypertension and may be important contributing factors to increased vascular resistance. Enhanced sensitivity to phorbol ester–induced PKC activation and the associated contractions have been observed in aortic rings from SHR²⁷ and from rats with aortic coarctation–induced hypertension.²⁸ Also, a PKC inhibitor attenuated contractile responses to norepinephrine and angiotensin II to a greater degree in aortic rings from SHR than in those from normotensive controls,²⁹ suggesting that contractile responses to these agonists have a greater dependence on PKC in SHR. Although the mechanisms by which PKC mediates vascular contraction are not fully elucidated, current evidence suggests that downstream effects of PKC activation to enhance calcium mobilization³⁰ and/or increase the sensitivity of contractile elements to calcium³¹ are important factors. Hence, it is plausible that the calcium-dependent tone in aortic rings from rats with aortic coarctation–induced hypertension is a consequence of increased PKC activity. Because PKC may participate in the mechanisms underlying myogenic responses to stretch,³² one may also consider the possibility that under the conditions of our study, the development of calcium- and PKC-dependent tone by the aortic smooth muscle of hypertensive rats is reflective of an exaggerated myogenic response to stretch of the aortic rings of these animals ex vivo.

Both calcium-dependent tone and staurosporine-inhibitable tone were greater in endothelium-denuded aortic rings than in endothelium-intact aortic rings from hypertensive rats. This finding suggests that an endothelium-derived factor inhibits the development of tone in aortic rings from hypertensive rats. Endothelium-dependent inhibition of the contractile actions of a variety of agonists is well documented. In our studies the endothelial factor appeared to be NO, because treatment of intact aortic rings from hypertensive rats with L-NAME to inhibit NO synthesis increased calcium-dependent tone. Therefore, it may be concluded that endogenous NO acts to suppress the development of calcium- and PKC-dependent tone in aortic rings from rats with aortic coarctation–induced hypertension. In strong support of this notion, SNP, acetylcholine, and L-arginine, but not D-arginine or L-lysine, reversed, in concentration-dependent fashion, the calcium- and PKC-dependent tone in aortic rings from these hypertensive rats. That the relaxant effects of SNP, acetylcholine, and L-arginine are due to augmentation of NO levels in aortic rings from hypertensive rats is corroborated by our observation that the relaxant effects of each of these agents were paralleled by increases in cGMP levels: SNP induced relaxation and increased cGMP in endothelium-intact and endothelium-denuded rings; acetylcholine induced relaxation and increased cGMP only in endothelium-intact rings; and L-arginine induced relaxation and increased cGMP in both endothelium-intact and endothelium-denuded rings.

L-Arginine–induced relaxation and increases of cGMP in aortic rings from hypertensive rats were blunted but not eliminated by rubbing to remove the vascular endothelium. This suggests that the inhibitory effect of L-arginine on isometric tension development in these aortic rings has both endothelium-dependent and endothelium-independent components. This finding parallels our previous report that contraction of aortic rings induced by inhibition of NO also has both endothelium-dependent and endothelium-independent components.⁴ Although we cannot unequivocally rule out the possibility that residual functional endothelium could account for the effects of L-arginine, this is unlikely because we have demonstrated that rubbing completely eliminates acetylcholine-induced relaxation⁴ and cGMP generation in aortic rings from rats with aortic coarctation–induced hypertension. The synthesis of NO is catalyzed by a constitutive NO synthase, found in endothelial cells,³³ and, in some pathological conditions, by inducible NO synthase found in vascular smooth muscle,³⁴ endothelial cells,³⁵ and activated macrophages³⁶ and leukocytes.³⁷ Thus, our data support the notion that inhibition of NO synthesis by L-NAME and augmentation of substrate availability to NO synthase with L-arginine at sites other than the vascular endothelium may account for the vasoactivity of these agents in endothelium-denuded aortic rings.

Our findings and conclusions conform with the hypothetical scheme illustrated in Fig 4. Aortic rings from hypertensive rats feature a high level of constrictor tone in the absence of exogenous vasoconstrictors. This tone is calcium-dependent and also appears to be related to a PKC-mediated mechanism of vascular contraction. Both exogenous and endogenous NO suppress the development of this tone in aortic rings from hypertensive rats. The actions of NO to antagonize the development of tone may be functional in nature or may, instead, involve more direct interactions with the constrictor mechanisms that are operational in the vasculature of hypertensive rats. Our findings raise the possibilities that a calcium-dependent, PKC-mediated mechanism of vascular contraction fosters elevation of vascular resistance in these rats with aortic coarctation–induced hypertension and that endogenous NO serves as a counterregulatory influence to this vasoconstrictor mechanism.

View larger version (12K): [in this window] [in a new window]   Figure 4. Schematic diagram shows the proposed effects of aberrant calcium metabolism and increased protein kinase C activity on vascular tone and the counterregulatory effect of nitric oxide.

	Acknowledgments

 
This work was supported by United States Public Health Service grants HL-18579, HL-35670, and HL-34300.

	Footnotes

 
Reprint requests to Alberto Nasjletti, MD, Department of Pharmacology, New York Medical College, Valhalla, NY 10595.

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