Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

Doubt has been raised about the expression of a functional Na+-K+-2Cl−cotransporter in rat skeletal muscle. In this study we present molecular and functional evidence for expression of a protein having the characteristics of a cotransporter. RT-PCR of RNA isolated from rat soleus muscle with primers to a conserved putative membrane-spanning domain resulted in a single product of predicted size. Sequencing of the product showed that it bears >90% homology with known rodent NKCC1 (BSC2) cotransporters. RNase protection assay of RNA isolated from the rat soleus muscle also identified this sequence. Immunologic detection of the cotransporter with two different antibodies indicated the presence of cotransporter protein, perhaps more than one, in blots of total muscle protein. Immunohistochemical detection by confocal microscopy localized the majority of expression of the protein to the muscle fibers. Functional studies of cotransport activity also indicate the appropriate sensitivity to inhibitors and ion dependence. Taken together, these data support the presence and function of Na+-K+-2Cl−cotransporter activity in the soleus muscle of the rat.

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.

Renin release was studied during the infusion of catecholamines (.7–5.6 µg/min) and angiotensin II (.25–1.05 µg/min) into the renal artery, and also during and following iv infusion of 16 µg/min noradrenaline. Filtration rate (Ccr), RPF, and RBF were estimated from the renal clearance of creatinine and the extraction ratio. Marked increases in renal vein renin titer and V-A renin difference occurred within 1 min of the onset of infusion of catecholamine into the renal artery and continued for 15 min beyond the infusion period. This infusion was always accompanied by diminished renal function. During iv infusion of noradrenaline, renin release did not usually occur, but on stopping the iv infusion, the ensuing fall in blood pressure was coincident with a marked release of renin apparently unaccompanied by decreased renal function. Infusion of angiotensin into the renal artery, in spite of causing renal vasoconstriction and marked decrease in urine volume, was not accompanied by renin release. The difficulties of measuring renin release under these conditions are discussed.

Small changes in sodium concentration [( Na]) are not generally considered to have a major direct effect on aldosterone secretion. However, a marked disruption in the renin-aldosterone relationship has been observed in a variety of hypernatremic and hyponatremic states. Therefore, we evaluated the hypothesis that small changes in [Na] have a potent direct effect on angiotensin II- and potassium-stimulated aldosterone secretion. The left adrenal gland, abdominal aorta, and surrounding periadrenal tissue were surgically isolated from mongrel dogs and perfused with Ringers bicarbonate solution at a pressure of approximately 57 mm Hg. Infusion of a KCl test solution at the beginning and end of most experiments produced similar increases in aldosterone secretion, thus documenting the stability of these preparations. After a stable response was established to either a low dose of angiotensin II or a small increase in perfusate [K], the [Na] was changed by adding or removing NaCl. Changing perfusate [Na] from 152 to 139 mM during the infusion of either angiotensin II or potassium caused 20- to 25-fold increases in aldosterone secretion. Increasing perfusate [Na] from 145 to 152 mM inhibited aldosterone secretion to a greater extent during stimulation by lower doses (40-50 pg/ml) than by higher doses (80-100 pg/ml) of angiotensin II. These data demonstrate that during moderate stimulation by angiotensin II or potassium, small changes in [Na] have a powerful inverse effect on aldosterone secretion by a direct action on the canine adrenal gland.

The purpose of these experiments was to determine if the powerful effect of sodium chloride concentration on angiotensin II- and potassium-stimulated aldosterone secretion by isolated perfused adrenal glands is mediated by the sodium or chloride ion or by the obligatory change in osmolality. We used isolated Ringer's bicarbonate perfused canine adrenal gland preparations to determine the effects of a variety of isosmotic, hyperosmotic, and hyposmotic solutions on angiotensin II- and potassium-stimulated aldosterone secretion. When we increased the osmolality of the perfusion medium (8-10 mosmol) by the addition of NaCl, sucrose, mannitol, or glucose, angiotensin II-stimulated aldosterone secretion was inhibited to a similar extent, whereas urea addition had no effect. Similarly, when we increased the osmolality of the perfusion medium (8-10 mosmol) by the addition of NaCl, sucrose, or mannitol, potassium-stimulated aldosterone secretion was also inhibited to a similar extent. In contrast to the increase in angiotensin II- and potassium-stimulated aldosterone secretion observed during hyposmotic reductions in NaCl concentration, (addition of sucrose) did not increase angiotensin II- or potassium-stimulated aldosterone secretion. Even the marked increase in aldosterone secretion caused by large hyposmotic reduction in NaCl concentration did not occur with an equivalent isosmotic reduction in NaCl concentration. These results clearly demonstrate that changes in NaCl concentration affect aldosterone secretion by a mechanism sensitive to the osmolality. Moreover, since hyperosmolality caused by urea addition had no effect on angiotensin II-stimulated aldosterone secretion, changes in intracellular volume or composition appear to be an important modulator of aldosterone secretion.

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