Jacquelyn M. Smith

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.

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.

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.

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.

Effect of parathyroid hormone on renin secretion.

Abstract The ability of parathyroid hormone (PTH) to increase renin secretion was investigated in pentobarbital-anesthetized dogs. An intravenous infusion of bovine PTH 1-34, at the dose of 0.028 μg kg-1 min-1 increased renin secretion by 149% (501 ± 105 to 1249 ± 309 ng hr-1 min-1); renin secretion returned to control values during the recovery period. In order to determine whether PTH acted directly on the kidney to increase renin secretion, PTH was infused into the right renal artery at doses of 0.0014 to 0.0028 μg kg-1 min-1 and renin secretion from the right kidney was compared to that from the left (control) kidney. Renin secretion from the right (PTH-infused) kidney was not greater than control values for that kidney or different from the renin secretory rate of the left (control) kidney. In contrast, the excretion rates of both phosphate and sodium from the right kidney were greater than control values and from the excretion rates of the left kidney. These data suggest that PTH, while acting directly on the kidney to increase phosphate and sodium excretion, does not elevate renin secretion by a direct renal action.

Altered Phospholipase Activities Related to α1-Adrenergic Receptor Supersensitivity of Aortas from Aldosterone-Salt Hypertensive Rats

Many of the concepts presented in this paper are summarized in Fig. 7. Some aspects are well supported while others are speculative. The operation of PLC in VSM is well established, and in some hypertensive models (AHR, SHRSP) PLC assays exhibited altered activation. Currently this pathway leading to the production of IP3 and DAG is considered to be the major regulator of Ca release from sarcoplasmic reticulum (SR) and Ca entry by channels (CaC). Regulation of PKC by [Ca]i and DAG is thought to play a major role in controlling Ca entry. PKC has also been proposed to regulate PLA2 as well as PLD in conjunction with elevated [Ca]i. An important issue to be resolved is whether receptor regulation of other lipases occurs independently of the PLC-[Ca]i-PKC axis. Currently information supporting receptor regulation is lacking for VSM, but few studies have been conducted. Our observation that NE stimulation of PLD activity occurs in VSM indicates that the control of VSM by biochemical messengers is much more complicated than previously proposed. This seemingly redundant pathway may allow VSM to use alternate substrates for producing PA and DAG than are readily available to PLC. It also allows PA to be produced directly without phosphorylation of DAG. Although the role of PA in the regulation of Ca entry was proposed earlier, definitive studies establishing this linkage are still required. Any PLD activity on PIP2 would produce biochemical messengers (PA, DAG) which could stimulate Ca entry without producing the messenger, IP3, associated with Ca release (inactive IP2 would be produced). If PLC and PLD were independently regulated by receptor-guanine nucleotide-regulatory protein (G-protein) complexes, this would offer the potential for some agonists to excite VSM by Ca release and Ca entry mechanisms while others may excite by Ca entry alone. This system would also circumvent the problem of limited substrate for cellular regulation of [Ca]i if PIP2 were the primary substrate. This limitation does not exist with other phospholipids such as phosphatidylcholine which is a preferred substrate for PLD. The presence of multiple phospholipases under separate receptor regulation allows for a wider range of tissue responses to various agonists, than a system which is linked only through the PLC-[Ca]i-PKC axis. The presence of a PLD pathway also reopens the interpretation of previous studies which demonstrated a resetting between receptor occupancy and production of second messengers by PLC.(ABSTRACT TRUNCATED AT 400 WORDS)

Potassium Turnover and Norepinephrine Sensitivity in the Thoracic Aorta of the Dahl Rat

Abstract This in vitro study evaluated the basal 42K turnover and response to norepinephrine (NE) in the thoracic aorta removed from Dahl salt-sensitive (S) and salt-resistant (R) rats. Five-week-old S and R rats were placed on either a high-salt (HS) or low-salt (LS) diet. After 5 weeks of the diet, systolic blood pressure, aortic weight/length ratio, and the cellular pool of K+ were elevated in the S-HS group only. In contrast, the steady state turnover of 42K, the NE ED50, and the response to a supramaximal dose of NE were the same in both groups of salt-sensitive and salt-resistant rats. These results suggest that, despite the presence of a greatly elevated systolic blood pressure and evidence of aortic hypertrophy, the intrinsic electrolyte metabolism of the vascular smooth muscle in the Dahl hypertensive rat is the same as that of the Dahl normotensive rat.

Nisoldipine inhibition of sodium influx into aorta from aldosterone-salt-hypertensive rats.

The purpose of this study was to determine whether increased sodium (Na) influx into the aorta was associated with aldosterone-salt hypertension in the rat and, if present, to determine what mechanisms contributed to the increase. Basal 24Na influx was elevated in aorta from the hypertensive rats (2.21+0.10 mmol/1 cell H2O/min, n = 25) compared with control-salt rats (1.75±0.04 mmol/1 cell H2O/min, n = 24). The calcium (Ca) antagonist nisoldipine inhibited the Na influx into aorta from hypertensive rats in a concentration-dependent manner. At 10 nM nisoldipine, the Na influx in hypertensive rats (1.52±0.14 mmol/1 cell H20/min, n = 10) was similar to control rats (1.66±0.18 mmol/1 cell H2O/min, n = l). The basal Na influx in aorta from hypertensive rats was not altered by dichlorobenzamil or ethylisopropylamiloride, selective inhibitors of Na-Ca and Na-H exchange, respectively. The Na influx was 2.21±0.10, 2.03±0.24, and 2.11 ±0.19 mmol/1 cell H2O/min for basal (n = 25), dichlorobenzamil (n = 4), and ethylisoproisopropylamiloride (n = ll), respectively. Inhibition of Na influx in hypertensive rats by 0.1 μM nisoldipine (ΔNa influx=−0.72±0.18 mmol/1 cell H2O/min, n = 9) was not significantly altered when applied with dichlorobenzamil (−0.72±0.21 mmol/1 cell H2O/min, n = 4) or ethylisopropylamiloride (−0.55±0.15 mmol/1 cell H2O/min, n = ll). These agents did not alter Na influx in control aorta. Our results suggest that, in aorta from aldosterone-salt-hypertensive rats, an elevated Na influx exists that is dependent on Ca entry through potential-operated Ca channels. Na-Ca and Na-H exchange do not appear to contribute significantly to the elevated Na influx, which is suggested to result from the activity of a Ca-dependent cationic channel.

Membrane transport in vascular smooth muscle and its relation to normal and altered excitation during hypertension

Evidence is presented for the operation of a Cl−Cl exchange diffusional transport system in rat aortic smooth muscle. The efflux of36Cl associated with this mechanism is doubled in rats made hypertensive with aldosterone-salt treatment. The residual efflux of Cl is similarly elevated in the hypertensives. This finding supports the hypothesis that increased membrane permeability to Cl is associated with aldosterone hypertension. The agonist-induced increases in aortic effluxes of42K exhibit a 10-fold reduction in the ED50 to norepinephrine in aldosterone hypertensive rats. The possible contributors to this supersensitivity were studied by means of a pharmacologic analysis of the action of competitive (phentolamine) and non-competitive (dibenamine) antagonism of norepinephrine induced increases in42K efflux. The dissociation constant for phentolamine, KB=1×10−8 M, was relatively unaltered in the hypertensive group, as was the dissociation constant for norepinephrine, KA=3.4−5.8×10−7 M. A substantial increase in receptor number and/or transduction efficiency (3–12 fold) was derived from the analysis. It is tentatively concluded that supersensitivity to norepinephrine during aldosterone hypertension may be more closely related to changes in receptor number and/or efficiency than in receptor affinity.