The interaction of vascular electrolytes and early spontaneous hypertension was studied in the rat aorta. Chemical composition (FLO, Na, K, Ca, Mg, Cl, collagen, and elastin), extracellular space, and cell water content were little changed. Only uronic acid and hexosaTnine contents were significantly elevated in the spontaneously hypertensive rat. Approximately 37% of the aortic weight was cellular. Functional changes in ion transport were observed in smooth muscle from hypertensive rats; the muscle exhibited decreased ability to accumulate K and extrude Na and increased turnover of -K exchange after adrenalectomy and reserpinization. The bioregulants, aldosterone, norepinephrine, and angiotensin had important actions on ion exchange. After adrenalectomy, aldosterone therapy reduced 4 -K exchange toward intact levels. Norepinephrine increased the rate of 42 K exchange with the dose-response relation having a lower median effective dose (ED S0 ) for spontaneously hypertensive rats (10-° g/ml) than it did for normal Wistar rats (2 X 10-" g/ml). Angiotensin also increased 42 K exchange with similar dose-response relations for both groups. I concluded that functional alterations observed in spontaneously hypertensive rats probably resulted from primary changes in ion transport by vascular smooth muscle rather than from secondary effects of altered regulatory systems. The decreased selectivity to K over Na and the increased turnover of ions could lead to increased reactivity to norepinephrine through effects on membrane potentials. This work was supported in part by U. S. Public Health Service Grants HE 07762 and HL 15852 from the National Heart and Lung Institute. KEYA preliminary report has appeared in abstract form (Fed Proc 31: 1020, 1972. Received April 4, 1973. Accepted for publication September 6, 1973. become available as a model for essential hypertension (3), but little change in the water, sodium, or potassium content of aortas from these rats has been observed during the early hypertensive phase (13 weeks old) (4). This observation raises the question of the role of vascular electrolyte metabolism in the pathogenesis of spontaneous hypertension and perhaps of other types of the disease.The objective of the present study was to reappraise the interaction of vascular electrolytes and spontaneous hypertension. Since ion transport by smooth muscle is especially important for regulating total wall contents (5, 6), the experimental approaches investigated functional properties as well as total wall composition. The influences of bioregulants were also studied to clarify points regarding hyperreactivity and to separate primary

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.

SUMMARY1. The water content, extracellular (60CoEDTA) space, ionic composition and ultrastructure of several mammalian smooth muscles were studied after incubation in solutions of varying ionic compositions and osmolarities.2. Substitution of KC1 for NaCl resulted in an increase in cell water, K and Cl, accompanied by little change in total wet weight. This was due to a reduction in the extracellular space.3. Changes in extracellular osmolarity produced a wider range of cell volumes in high KCl solutions than in Krebs. The addition of 29-58 mm sucrose to high KC1 prevented the swelling.4. Electron microscopy of smooth muscle swollen in high KCl solution revealed light (less electron opaque than normal) fibres of increased diameter, reduction in extracellular space, and nuclear swelling. The normal thick filament lattice was destroyed in swollen, osmium-fixed smooth muscles.5. The ultrastructural changes ascribed to swelling were absent in smooth muscles, (a) depolarized in high K2SO4 solutions, (b) in high KCl solutions with 29-58 mM sucrose, and (c) returned to normal Krebs solution for recovery from swelling.6. Smooth muscles incubated in high KCl (swollen) and high K2SO4(unswollen) exhibited similar contractile responses, suggesting the filament lattice was intact until fixation, and that the contractile mechanism can operate over a relatively wide range of actin to myosin separations. 8. The cell water of fixed taenia coli was reduced (a) by incubation in hypertonic solution followed by fixation in normal glutaraldehyde, or (b) by fixation of normal tissue in hypertonic glutaraldehyde. Osmotic responses during aldehyde fixation may be a source of artifact in the visualization of the normal filament lattice.

1. [(60)Co]EDTA has been evaluated as an extracellular marker in guinea-pig taenia coli and rabbit myometrium.2. The complex was found to be stable, non-toxic, convenient to use and had the advantage of being readily measured without interfering with the analysis of water and electrolyte contents.3. In both smooth muscles [(60)Co]EDTA distributed rapidly into a volume equivalent to that available to [(14)C]sorbitol and slightly greater than that available to inulin.4. The optimum concentrations of carrier required to effectively saturate any adsorption sites were 0.1 mM-CoEDTA for taenia coli and 1.0 mM for myometrium.5. [(60)Co]EDTA was distributed into a volume equivalent to the total water content of rabbit tendon, indicating that it was not excluded from connective tissue water.6. About 5% of the [(60)Co]EDTA taken up by smooth muscle in 15 minutes exhibited slow efflux kinetics. This property was also exhibited by [(14)C]sorbitol and has been reported for other extracellular markers.7. The efflux of 95% of [(60)Co]EDTA followed bulk diffusion kinetics for both taenia coli and myometrium.8. The simultaneous efflux of (24)Na indicated that 1-2% exchanged slowly while 98-99% of the (24)Na exchange could be described by bulk diffusion kinetics, similar to [(60)Co]EDTA. About 15% of this (24)Na was contained outside of the [(60)Co]EDTA space.9. More detailed studies involving the analysis of simultaneous efflux of [(60)Co]EDTA and (24)Na may help to locate and characterize the properties of the excess rapidly exchanging sodium (12-15 m-equiv/kg. wet wt.) in smooth muscle.

Change in aortic water and electrolyte distribution and in ion turnover were studied during the development of deoxycorticosterone acetate (DOCA) hypertension in the rat. Treatment with DOCA plus saline during the prehypertensive phase was associated with increases in 42K turnover (0.0142 +/- 0.0005 vs. 0.0102 +/- 0.0003 min-1), cell water (0.89 +/- 0.03 vs 0.76 +/- 0.02 kg/kg dry weight), and the ratio of weight to length. These parameters were further increased during the development of hypertension. Significant increases were also observed in total K, Ca, and Mg contents and in Na and C1 contents corrected for the extracellular space. The turnover of 36Cl was increased (0.230 +/- 0.006 vs. 0.136 +/- 0.004 min-1) in DOCA hypertensive rats as was the content of slowly exchanging Cl. Removal of extracellular Ca greatly increased the steady-state turnover of 42K. For control rats, a Ca concentration of 0.1 mM reduced the rate of 42K turnover to less than 50% of the Ca-free value (0.063 +/- 0.004 min-1), whereas DOCA hypertensive rats exhibited only a 10% reduction. At the highest Ca concentration, 5 mM, the 42K turnover was greater in DOCA-treated rats with the hypertensives operating at 67% of maximum efflux or about twice the efflux in controls. It is concluded that significant alterations in ion transport by vascular smooth muscle occur before and during the development of hypertension induced by treatment with DOCA plus saline. Such changes may result from a reduced ability of Ca to stabilize the membrane. It is proposed that such alterations contribute to the changes in vascular reactivity and the hypertrophy associated with hypertension.

No abstract

Heme oxygenase-1 knockout, H mox

We tested the hypothesis that exercise training reduces the sensitivity of coronary smooth muscle to endothelin-1 (ET-1), with the adaptation being greater in male than in female miniature swine. The efficacy of training was similar in males and females. Cumulative ET-1 contractile responses of coronary branches and left circumflex artery were significantly shifted to the right in exercise-trained (Ex) males but not in Ex females. Analyses of the excitatory concentration causing a 50% response (EC(50)) showed a 1.7- to 2.2-fold shift in Ex males with no change in maximum tension. Nonselective blockade of K-channel activity with tetraethylammonium (TEA; 30-50 mM) significantly shifted the EC(50) to a lower concentration in both Ex males (1.25-fold) and Ex females (2.2-fold) but not in sedentary (Sed) groups. Females (combined Sed and Ex) exhibited a greater response to TEA than did combined Sed and Ex males. Changes in [(32)P]phosphatidic acid ([(32)P]PA) provided an indicator of ET-1-induced phospholipase activity. The magnitude of the [(32)P]PA response was reduced by Ex in both males and females without affecting the EC(50). It is concluded that the contractile sensitivity of coronary arteries to ET-1 is influenced by physical activity in a gender-dependent manner. It is unclear why the contractile sensitivity in females was not reduced by Ex as in the males, because Ex significantly affected responses to TEA and ET-1 stimulation of [(32)P]PA production in both males and females. A potential gender difference in K-channel function may contribute to this discrepancy.