Allan W. Jones

Altered Ion Transport in Vascular Smooth Muscle from Spontaneously Hypertensive Rats: Influences of Aldosterone, Norepinephrine, and Angiotensin

The interaction of vascular electrolytes and early spontaneous hypertension was studied in the rat aorta. Chemical composition (H2O, Na, K, Ca, Mg, Cl, collagen, and elastin), extracellular space, and cell water content were little changed. Only uronic acid and hexosamine 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 42K (0.0165 ± 0.0009 vs. 0.0086 ± 0.0002 min−1) and 36C1 (0.162 ± 0.011 vs. 0.118 ± 0.003 min−1). Spontaneously hypertensive rats maintained increased 42K exchange after adrenalectomy and reserpinization. The bioregulants, aldosterone, norepinephrine, and angiotensin had important actions on ion exchange. After adrenalectomy, aldosterone therapy reduced 42K exchange toward intact levels. Norepinephrine increased the rate of 42K exchange with the dose-response relation having a lower median effective dose (ED50) for spontaneously hypertensive rats (10−9 g/ml) than it did for normal Wistar rats (2 × 10−9 g/ml). Angiotensin also increased 42K 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.

Altered ion transport in aortic smooth muscle during deoxycorticosterone acetate hypertension in the rat.

Changes 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 “K turnover (0.0142 =t 0.0005 vs. 0.0102 ± 0.0003 min1”), 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 36C1 was increased (0.230 ± 0.006 vs. 0.136 ± 0.004 min−1) in DOCA hypertensive rats as was the content of slowly exchanging C1. Removal of extracellular Ca greatly increased the steady-state turnover of “K. For control rats, a Ca concentration of 0.1 DIM 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 IDM, the “K 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.

Comparison of Rubidium-86 and Potassium-42 Fluxes in Rat Aorta

Potassium transport has frequently been assessed by measurement of 86Rb. However, recent reports indicated that the K ion channels are selective to K over Rb. Therefore, the purpose of this study was to evaluate whether the basal and stimulated fluxes of Rb and K were equivalent in rat aorta in the absence of endothelium. The ouabain-sensitive (active) uptake of 86Rb and 42K was similar. However, the basal 86Rb efflux was only 80% of the 42K efflux. Norepinephrine and KCl depolarization stimulated 86Rb and 42K effluxes via a calcium-dependent process. The stimulated 86Rb efflux ranged from 56 to 74% of the 42K efflux. Diltiazem reduced the KCl-stimulated 86Rb and 42K effluxes. The 86Rb efflux was 82% of the 42K efflux in the presence of KCl plus diltiazem, similar to that under basal conditions. Substitution of Rb for K in the incubation solution was associated with a marked increase in spontaneous contractile activity. There was no change in the norepinephrine concentration required for a 50% stimulation of contraction or 86Rb efflux from Rb-loaded tissues. We conclude from these studies that the basal and calcium-activated potassium channels are selective for K over Rb and therefore 86Rb fluxes quantitatively underestimate that of 42K. However, 86Rb is an appropriate substitute for the measurement of active K transport.

Heme oxygenase-1 deficiency leads to alteration of soluble guanylate cyclase redox regulation

Heme oxygenase-1 knockout, Hmox1(−/−), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in Hmox1(−/−) mice. The contractile responses [superior mesenteric arteries (SMA), from female Hmox1(−/−) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in Hmox1(−/−) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in Hmox1(−/−) than Hmox1(+/+) mice (p < 0.001). Branches from Hmox1(−/−) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4′-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. Hmox1(−/−) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were Hmox1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of Hmox1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of Hmox1(−/−) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1and β1 subunit levels were reduced to <50% of Hmox1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of Hmox1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.

Calcium-dependent fluxes of potassium-42 and chloride-36 during norepinephrine activation of rat aorta.

This study was designed to determine whether α receptor-stimulated monovalent ionic fluxes in rat aorta required calcium, and, if so, whether both extracellular calcium and cellularly stored calcium are active. Calcium removal in the presence of 10 mM magnesium (to maintain membrane stability) inhibited the norepinephrine-stimulated increase in potassium-42 and chloride-36 efflux. However, the norepinephrine-stimulated increase in sodium-24 influx was relatively resistant to calcium depletion. Protocols were designed to measure the time course for the changes in potassium-42 efflux and contraction when calcium was removed or replaced in the presence of norepinephrine. The dose-dependent effect of a calcium antagonist (diltiazem) was also measured. A close correlation (r = 0.94) was found between inhibition of contraction and potassium-42 effluxes which followed the regression: % potassium-42 response = 1.0 × (% contraction) + 1.8%). The slope of 1.0 and intercept near zero suggests the hypothesis that norepinephrine-stimulated potassium-42 efflux and contraction are codependent on cellular calcium concentration. This co-dependence held for short phasic responses (± 1 minute), as well as longer tonic responses (>5 minutes). It appears that calcium-dependent potassium-42 effluxes can be supported by both the influx of extracellular calcium and release of cellular stores. It is concluded that calcium-dependent potassium channels (and possibly chloride channels) are operative in rat aorta and are an important component of the graded membrane response to norepinephrine. The sodium channels, however, do not appear to share this same calcium dependency.

Increased barium influx and potassium current in stroke-prone spontaneously hypertensive rats.

Arterial potassium permeability is increased in hypertension. In this study we conducted voltage-clamp experiments to determine whether the whole-cell K+ current is increased in a Ca(2+)-dependent manner in aortic smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSP). Aortic cells from Wistar-Kyoto (WKY) rats and SHRSP demonstrated an outward rectifying current elicited by depolarization. The current was carried primarily by K+, because intracellular Cs+ replacement eliminated more than 97% of the current. The current density was higher (P < .05) in SHRSP cells at positive potentials. In the presence of LaCl3 (200 mumol/L) or tetraethylammonium (10 mmol/L), the residual current was similar in WKY and SHRSP cells. Also, the current density did not differ between WKY and SHRSP cells in which the intracellular Ca2+ concentration was clamped at zero. Fura 2 ratio measurement showed similar resting myoplasmic Ca2+ concentration (Ca2+m) in WKY and SHRSP cells (100 +/- 10 versus 117 +/- 9 nmol/L, P = .2). Under low extracellular Na+ conditions, which had a minimal effect on Ca2+m, Ba2+ replacement of Ca2+ caused a continuous and approximately linear increase in the fura 2 ratio, which was twofold faster in SHRSP cells. Because Ca2+ pumps do not transport Ba2+ and Na(+)-Ca2+ exchange was inhibited by low extracellular Na+, this increase reflected unidirectional Ba2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered biochemical and functional responses in aorta from hypertensive rats.

Factors that lead to supersensitivity of vascular smooth muscle to norepinephrine during aldosterone-salt-induced hypertension in rats appear to reside beyond ligand-α-adrenergic receptor binding, which we have shown previously to be normal. The objective of this study was to determine whether significant shifts occur in the coupling between receptors and the production of putative second messengers. Measures of [3H]myo-inositol phosphates in aorta (endothelium removed) exhibited a concentration-dependent increase to norepinephrine, with the 50% response shifted significantly to the left in the hypertensive group (7.0 ± 0.9 × 10−7 M in 8 control rats vs 1.1 ± 0.2 × 10−7 M in 8 hypertensive rats; p < 0.001). The production of [32P]phosphatidic acid was also shifted (6.5 ± 2.5 × 10−7 M in 16 control vs 1.9 ± 0.8 × 10−7 M in 12 hypertensive rats; p < 0.05). The functional responses of 42K efflux and contraction to norepinephrine were also significantly shifted threefold to 15-fold in the hypertensive group (p < 0.001), but the 50% response typically occurred at a 10 to 100 times lower concentration than that for the production of mvo-inositol phosphates and phosphatidic acid. The amplification between receptor occupancy and functional responses apparently occurs beyond the production of phosphoinositide metabolites. The fivefold shift in the 50% response of biochemical end points for the hypertensive group accounted for most of the shift (sixfold) in the functional end points. It is concluded that the increased efficacy in the hypertensive group resulted more from shifts in the relation between receptor occupancy and production of phosphoinositide metabolites than from shifts in the action of these metabolites on functions that control 42K efflux and contraction.

Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion-Induced Leukocyte-Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+-activated K+ Channels

Please cite this paper as: Wang, Kalogeris, Wang, Jones and Korthuis (2010). Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion‐Induced Leukocyte‐Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+‐activated K+ Channels. Microcirculation17(6), 427–438.

Isoform-selective 5'-AMP-activated protein kinase-dependent preconditioning mechanisms to prevent postischemic leukocyte-endothelial cell adhesive interactions

We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.

Calcium antagonists inhibit elevated potassium efflux from aorta of aldosterone-salt hypertensive rats.

The purpose of this study was to evaluate the effect of calcium antagonists on basal tension and the elevated 42K efflux in aorta from aldosterone-salt hypertensive rats. Diltiazem decreased the basal tension (2.0 ±0.4 g) as well as the phasic contractile activity and returned the elevated 42K efflux (0.018±0.002/min) toward control values (0.010±0.001/min, p < 0.001). The diltiazem median inhibitory concentration (IC50) for basal tension (0.04±0.02 juM), however, was sevenfold less than the IC50 for basal 42K efflux (0.22 ±0.08 /tM, p < 0.01). The basal 45Ca influx in aorta from aldosterone-salt hypertensive rats (120±4 /JJVI/1 cell H2O/min) was also decreased by diltiazem in a concentration-dependent manner, whereas the 45Ca influx in aorta from control-salt rats (135±3 /JM/1 cell H2O/min) was not altered. Similarly, the dihydropyridine nisoldipine eliminated the basal tension (2.7±0.5 g) and returned the elevated basal 42K efflux from the hypertensive aorta toward control levels (0.010 ±0.0003/min, p< 0.001). The nisoldipine IC50 for basal tension (0.016±0.01 nM) was 160-fold less than the IC50 for basal 42K efflux (1.8±1.2 nm,/;<0.001). Neither diltiazem nor nisoldipine altered the basal 42K efflux or contractile activity of aorta from control-salt rats. These results suggest that the basal tension and elevated 42K efflux in aorta from aldosterone-salt hypertensive rats are supported by the entry of extracellular calcium into the tissue through potential-operated calcium channels.