Changes in sodium appetite in cattle induced by changes in CSF sodium concentration and osmolality

Alteration of the sodium concentration in the cerebrospinal fluid (CSF) of sheep induces reciprocal changes in sodium appetite. Similar studies have now been performed in cattle. Heifers were prepared with a unilateral parotid fistula and guide tubes were implanted in the skull for the introduction of probes into the lateral ventricles in order to sample CSF and infuse artificial CSF solutions. The cows were Na depleted by loss of saliva for 46 hr and then given free access for 2 hr to 300 mM NaCl/NaHCO3 solution. Artificial CSF infusions at 1.9 ml/hr were begun one hour before Na access. In control experiments, the cows drank 26.4 +/- 1.2 l of Na solution in 2 hr, 1.2 +/- 0.2 l of water in the preceding hour, and 0.3 +/- 0.1 l of water during Na access. Sham or standard isotonic CSF infusions did not alter these values. CSF [Na+] rose from approximately 142 to approximately 148 mmol/l, attributable to the effects of drinking the large volume of hypertonic Na solution. Infusion of 500 mM NaCl CSF increased CSF [Na+] and reduced Na intake and increased water intake. Infusion of 700 mM mannitol: 150 mM NaCl CSF reduced CSF [Na+] and increased both Na and water intake. Infusion of a mixture of these solutions had no effect on CSF [Na+] and increased water intake only. Infusion of 270 mM mannitol CSF reduced CSF [Na+] and slightly reduced Na intake. Standard isotonic CSF containing 0.5 or 2.0 micrograms/ml of angiotensin II increased water intake only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for cerebral sodium sensors involved in water drinking in sheep

Evidence supporting cerebral Na sensors in the initiation of thirst is the greater water drinking which occurs with intracerebroventricular (ICV) infusion of hypertonic NaCl than with ICV hypertonic saccharide solution. However, ICV infusion of hypertonic saccharide solution causes a reduction in CSF [Na], even though the saccharide is made in solution with normal [Na] of 150 mM. To prevent this, ICV infusion of hypertonic sucrose in high Na solution was made. The ICV infusion of 0.3 M sucrose/0.3 M NaCl-CSF caused water intake of 416 ± 173 ml (mean ± SEM) in 6 sheep, and CSF [Na] was 151 ± 0.9 mM, but ICV infusion of equiosmolar 0.45 M NaCl-CSF caused greater water intake of 1097 ± 202 ml and CSF [Na] increased to 178.8 ± 3.6 mM. Control ICV isotonic CSF did not cause drinking and CSF [Na] was 150.5 ± 0.8 mM whereas ICV 0.6 M sucrose-CSF ([Na]=150 mM) caused water intake of 132 ± 63 ml with CSF [Na] falling to 139.3 ± 1.3 mM. These results indicate specific brain NaCl sensors involved in thirst. Osmoreceptors may also exist because some drinking occurred with ICV sucrose despite reduced CSF [Na].     

Influence of mannitol-induced reduction in CSF Na on nervous and endocrine mechanisms involved in the control of fluid balance

Infusions (20 μl/min) of isotonic (0.27 M) and hypertonic (0.7 M) mannitol dissolved in Na-free artificial CSF were made for 1 h. into the lateral cerebral ventricle (IVT) of conscious water-replete sheep. The IVT infusion of both 0.27 M and 0.7 M mannitol induced a water-diuresis. Samples of CSF were collected prior to, and 5, 35, 65 and 125 min after the end of the infusion. These consistently showed a reduction in CSF [Na], while CSF osmolality remained unchanged after 0.27 M mannitol, and was considerably increased after 0.7 M mannitol. In the 44 h dehydrated sheep IVT infusion of 0.7 mannitol in Na-free artificial CSF was made for 6 h. The water deprivation as such caused a marked increase in plasma and CSF [Na] and osmolality. The 6 h IVT infusion of hypertonic mannitol further increased the CSF osmolality, while CSF [Na] decreased and reached a value below the normal for water-replete animals. The infusion also induced a fall in plasma ADH resulting in a water-diuresis, and extinguished the thirst of the dehydrated sheep. Furthermore, the infusion markedly reduced renal sodium excretion without causing any substantial change in blood aldosterone, in spite of the fact that there was a conspicuous increase in plasma renin concentration. The study supports the view that sodium sensitive receptors close to the cerebral ventricular system participate in the regulation of ADH secretion, water intake, renin release, and renal sodium excretion.     

External calcium sensitivity of low sodium contractures in the control and hypertrophied right ventricle of the ferret.

The existence of possible differences of calcium (Ca2+) fluxes through the sarcolemmal sodium-calcium (Na+/Ca2+) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture--as an indicator of the intracellular Ca2+ concentration--induced by partial or total withdrawal of external sodium (Na+), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca2+ concentration ([Ca2+]o) of 3 mM, the dependence of the contractures on extracellular sodium concentration ([Na+]o), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca2+]o and contracture amplitude at various [Na+]o showed that the apparent affinity of the contracture for [Ca2+]o was decreased in hypertrophied preparations. Thus, in 0 mM [Na+]o, half-maximal contracture was induced at a [Ca2+]o of 0.012 +/- 0.016 mM and 0.171 +/- 0.021 mM in control (n = 11) and hypertrophy (n = 12) respectively (P less than 0.001). Although these data may be indicative of a decreased Ca2+ influx through the Na+/Ca2+ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na+]o withdrawal.     

Increase in K+ and alpha-AIB active transport in CHO cells after low [K+] treatment

We have studied the effects of prolonged incubation in low [K+] medium (approximately 0.3 mM) on both K+ and amino acid transport in Chinese hamster ovary (CHO) cells. When incubated in low [K+] medium, CHO cells redressed partially the loss of intracellular K+ after 12 h. After 24 h of incubation, both the activity of Na+-K+-ATPase in crude homogenates, and the transport capacity (Vmax) for ouabain-sensitive (i.e., active) K+ influx approximately doubled. The magnitude of the ouabain-insensitive (i.e., passive) K+ influx decreased by 50%. Thus the regulatory response involves an apparent increase in Na+-K+ pump and a decrease in K+ leak. The transport capacity for the nonmetabolized amino acid, alpha-aminoisobutyric acid (alpha-AIB), also increased after 24 h in low [K+] medium. The Vmax for the Na+-dependent (i.e., active) alpha-AIB influx increased by about 150%, and the magnitude of the Na+-independent influx increased by 20-40%. These changes in alpha-AIB transport result in a twofold greater capacity to accumulate this amino acid. Thus the regulation of K+ and alpha-AIB transport systems appears to be linked and possible mechanisms of this linkage are discussed.     

31P and 23Na NMR spectroscopy of normal and ischemic rat skeletal muscle. Use of a shift reagent in vivo

23Na NMR spectroscopy was used 1, to define the distribution of the shift reagent for cations, triethylenetetraminehexaacetatedysprosium(III), DyTTHA3-, in the living rat; 2, to define the characteristics of the Na resonances reporting intra- and extracellular Na+ in skeletal muscle in vivo; and 3, to calculate the Na+ concentrations in the intra- and extracellular spaces of the gastrocnemius muscle during well-perfused and ischemic conditions. The concentration of DyTTHA3- infused intravenously into the jugular vein of the living rat reached a maximum value of 8-9 mM in the extracellular space of the muscle after ca 40 min of infusion. This allowed excellent discrimination of extra- and intracellular Na signals (Nao and Nai, respectively) and did not spoil the resolution of concurrent 31P NMR spectra. Infusion of shift reagent changed neither hemodynamic performance of the rat nor the high-energy phosphate content of skeletal muscle. Shift reagent enters ca 15% (v/w) of the rat body weight; this corresponds to almost all of the "fast" or rapidly permeable extracellular space. It is excreted from the body with a pseudo-first order rate constant of 0.0158 min-1. In resting muscle, we estimate that [Na+]i is 3-5 mM and, in muscle perfused with the sodium salt of the shift reagent, that [Na+]o in the fast exchangeable extracellular space is 166 mM. During 11 h of ischemia at 37 degrees C, the area of the Nai+ signal area monotonically increased sixfold. Based on estimates for maximum changes in fluid shifts reported by the decrease in the area of the Nao signal area, we calculate that the lower limit for [Na+]i after 11 h of ischemia is 27 mM. The NMR-visibility factors for the extracellular and intracellular Na+ signals are essentially the same. This study demonstrates that the shift reagent DyTTHA3- is acutely non-toxic and that the 23Na NMR spectra obtained can be used to quantitate [Na+]o and [Na+]i in tissues in vivo. Using this technique, we found that the transmembrane sodium gradient fell from ca 35 in well-perfused skeletal muscle to less than 6 during prolonged ischemia.     

Anomalous potential difference responses to changes in sodium concentration in the antrum of frog stomach

Previously, an electrogenic Na-Cl symport was found in the fundus, and the question arises of whether there is one in the antrum, a tissue that does not secrete acid. In an in vitro preparation of the antrum of Rana catesbeiana, we found that when the [Na+] in nutrient solution was decreased (choline for Na+) the transmucosal potential difference (PD) decreased (the positivity of nutrient side decreased), and when the [Na+] was increased the PD increased. These PD changes were anomalous for Na+ but not for choline. A linear relationship for PD versus log [Na+] and not versus log [choline] excluded a choline conductive pathway. The anomalous PD response was decreased but not abolished by 10(-3) M ouabain. Normal PD responses resulted from [Na+] changes in Cl--free (SO2-4) solutions. PD responses to changes in nutrient [Cl-] were normal but decreased in the absence of Na+. Data are compatible with a passive electrogenic Na-Cl symport with more chloride than sodium ions transported per cycle, as in the fundus. Symport conductance-to-total Cl- conductance ratio is higher in antrum than in the fundus. Data from the luminal side were compatible with apical membrane conductances for Na+ and Cl-.     

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system.

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.     

Ion-osmotic hyperthermia during exercise in dogs.

The influence of intravenous infusions of various concentrations of NaCl solutions on temperature regulation was investigated in dogs at rest and during moderate exercise for 1 h on a treadmill. Infusion of hypertonic solutions either before and during exercise resulted in elevated (P less than 0.05) plasma Na+ and osmotic concentrations and produced higher equilibrium levels (P less than 0.05) of rectal temperature (Tre) during exercise (prehypertonic 40.9 degrees C vs. no infusion 40.4 degrees C; hypertonic 40.8 degrees C vs. isotonic infusion 40.4 degrees C), but not at rest. Increasing the [Na+] and osmotic concentrations above 170 meq/liter and 325 mosmol/kg, respectively, resulted in no additional increase in exercise Tre. Water consumption during exercise decreased (P less than 0.05) plasma [Na+], osmolality, and the equilibrium level of Tre to control levels. There was no effect of changes in plasma volume (PV) of +/- 8% on the time course, equilibrium level, or change in Tre during exercise. At the end of exercise, there were moderate correlations (P less than 0.01) between Tre and [Na+] (r = 0.51) and Tre and osmoti (r = 0.52) concentrations. It was concluded that a) the exercise Tre responses of the dog respond quantitatively like man to elevated plasma [Na+] and osmolality, b) the Tre levels are not influenced by changes in PV, and c) water intake significantly reduces the ion-osmotic hyperthermia.     

Possible involvement of sodium pump in the relaxation of rat aorta induced by alpha-human atrial natriuretic polypeptide

In order to clarify whether the sodium handling of smooth muscle is associated with the relaxing action of alpha-human atrial natriuretic polypeptide (alpha-hANP), we examined the sodium pump-related effects of alpha-hANP on rat aortic smooth muscles. Application of Ca2+ (1.0 to 10.0 mM) to the muscle preincubated in Ca2+-free, and K+-free or 0.5 mM K+ medium for 60 min induced a contraction. Pretreatment with alpha-hANP (1 x 10(-8) M) decreased the contraction evoked in 0.5 mM [K+]o but not that in K+-free medium. After a contraction was elicited by norepinephrine in K+-free solution, an addition of KCl (1.4-5.4 mM) caused a transient relaxation in a concentration-dependent manner, presumably due to the activation of electrogenic Na pump. The alpha-hANP enhanced the relaxation, which was sensitive to ouabain, and the potentiation by alpha-hANP was inversely related to the concentration of K+ added. When alpha-hANP was applied to relax the muscle precontracted by norepinephrine in the varied concentration of external K+, alpha-hANP-induced relaxation was greater in 1.4 or 2.7 mM [K+]o than in 0 or 5.4 mM [K+]o. These results suggest that the vasodilating effect of alpha-hANP is at least partially mediated by the activation of electrogenic Na, K-pump and this effect is prominent when the Na, K-pump is partially suppressed.     

Increased resistance to haemorrhage induced by intracerebroventricular infusion of hypertonic NaCl in conscious sheep.

The effect of elevated cerebrospinal fluid Na+ concentration (CSF [Na+]) on the tolerance of blood loss, and concomitant cardiovascular and humoral responses were studied in conscious sheep. A slow (0.7 ml kg-1 min-1) venous haemorrhage was continued until the mean systemic arterial pressure suddenly decreased to less than 50 mmHg, or in the absence of hypotension, until a total blood loss of 25 ml kg-1. Significantly more blood had to be removed to induce hypotension in animals receiving an intracerebroventricular (i.c.v.) infusion (0.02 ml min-1) of 0.5 M NaCl (starting 30 min before haemorrhage and continued throughout the experiment) compared to control haemorrhages without concomitant i.c.v. infusion (22.7 +/- 1.2 ml vs 16.9 +/- 0.9 ml kg-1). In one animal, subjected to 0.5 M NaCl infusion, the blood pressure was still maintained at 25 ml kg-1 of haemorrhage. In spite of a larger blood loss, animals receiving i.c.v. infusion of hypertonic NaCl had an improved recovery of the blood pressure after haemorrhage, due to a better maintained cardiac output rather than to a reinforced increase of the vascular resistance. The improved cardiovascular responses to haemorrhage during elevated CSF [Na+] are not readily explained by the effects on the plasma concentrations of vasopressin, angiotensin II or noradrenaline, although the latter was augmented. The plasma protein concentration decreased already during the 30 min of hypertonic NaCl infusion preceding haemorrhage, and the haemodilution caused by the subsequent blood removal was aggravated, which indicates that this treatment also causes transfer of fluid to the plasma compartment. We conclude that elevated CSF [Na+] increases tolerance to haemorrhage and improves cardiovascular function after blood loss in sheep. Since the haemodynamic responses in many respects were similar to those reported in response to the systemic administration of a small volume of hypertonic NaCl solution in haemorrhagic shock, part of the effect of that treatment may be mediated via cerebral effects of increased Na+ concentration.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

On the importance of CSF Na in the regulation of renal sodium excretion and renin release

Infusions (20 microliters/min) of isotonic (0.27 M) mannitol dissolved in Na-free artificial cerebrospinal fluid (CSF) were made for 2 h into the lateral cerebral ventricle (IVT) of conscious 68 h dehydrated sheep. The IVT infusion induced a conspicuous drop in renal sodium excretion and marked rise in plasma renin concentration (PRC). The antinatriuretic response to the IVT infusion was not altered by the intravenous administration of ADH or te converting enzyme blocker (SQ 14225, Captopril). Surgical bilateral renal denervation did not change the antinatriuretic response while the increase in PRC was extinguished. Samples of CSF were collected prior to, and 15 min after the end of the infusion. These showed a reduction in CSF [Na], while CSF osmolality remained unchanged. The study supports the view that sodium sensitive receptors close to the cerebral ventricular system participate in the regulation of renal sodium excretion and renin release, it also suggests that renal sodium excretion is affected by an unknown hormonal factor of cerebral origin, while the release of renin seen in response to a reduction in CSF [Na] is mediated by the renal nerves.     

Significance of extracellular potassium in central respiratory control studied in the isolated brainstem-spinal cord preparation of the neonatal rat

The significance of extracellular potassium in central respiratory control was investigated using the isolated brainstem-spinal cord preparation of the neonatal rat. Depth profiles of extracellular potassium activity ([K+])ECF in the medulla were measured with ion-sensitive microelectrodes. Although [K+]ECF increased with depth in medullary tissue during control (4 mM) and low (1 mM) potassium concentration ([K+])CSF superfusion, this gradient disappeared with higher [K+]CSF. With low [K+]CSF (1 mM), respiratory CO2 responsiveness was abolished, and increased with high [K+]CSF (8 mM). Respiratory frequency (fR) was diminished at low [K+]CSF (1 mM), and increased with elevated [K+]CSF (8 and 16 mM); with yet higher [K+]CSF (32 mM) apnea occurred after a transient increase in fR. Perforated patch recording revealed that high [K+]ECF decreased membrane resistance, depolarized membrane potential, and increased firing frequency in most of the recorded medullary neurons. High [K+]ECF also increased excitatory and inhibitory post-synaptic potentials of medullary neurons and augmented the functional connectivity among neurons. It is concluded that [K+]ECF is of importance in the maintenance of respiratory rhythm and central chemosensitivity.     

Lowered cerebrospinal fluid sodium antagonizes effect of raised blood sodium on salt appetite

Moderately Na-deficient sheep (i.e., Na deficit = 300-400 mmol) will correct their deficit when given hypertonic NaHCO3 solution to drink. Access to NaHCO3 was provided by bar press for 2 h only each day following 22 h of salivary loss from a parotid fistula. Each delivery by bar press provided 9 mmol of NaHCO3 and, of the 46.3 +/- 2.5 deliveries made and drunk in 2 h, 80-90% were made in the first 20 min. Ten minutes before access to NaHCO3 commenced an intracarotid infusion of 4 M NaCl at 1.6 ml/min for 30 min was initiated. This infusion reduced intake by approximately 80% and increased both plasma and cerebrospinal fluid sodium concentration (CSF[Na]). Intraventricular (ivt) infusion of 0.7 M mannitol in artificial CSF at 1 ml/h for 3 h begun 1 h before access to Na by bar press lowered CSF[Na] and approximately doubled voluntary Na intake. The combination of the two procedures resulted in NaHCO3 intake similar to base line. That is, the ivt infusion of 0.7 M mannitol counteracted the inhibition of Na appetite produced by the systemic infusion of hypertonic NaCl, and this was associated with attenuation of the effect of the systemic 4 M NaCl infusion on CSF[Na]. The results suggest that the effects of both the ivt and the systemic infusions are mediated via the same sensor system located within the neuropil.     

Inhibition of vasopressin-release during developing hypernatremia and plasma hyperosmolality: an effect of intracerebroventricular glycerol

In non-hydrated goats prolonged (3 h, 0.02 ml/min) intracerebroventricular (IVT) infusion of 0.35 M glycerol depressed the plasma vasopressin level during the entire infusion period which resulted in a conspicuous water diuresis outlasting the infusion by about 20 min. Since no compensatory drinking occurred during this sustained water diuresis it gradually induced pronounced dehydration (loss of greater than 1 liter of total body water causing 5% increase in plasma [Na+] and osmolality). The same degree of dehydration was in other experiments induced by water deprivation. It then caused a 5-fold increase in plasma vasopressin level. Corresponding IVT infusions of 0.35 M d-glucose depressed plasma vasopressin level only during the first half of the 3 h infusion period. Consequently, the resulting water diuresis was transient and subsided before the glucose infusion was finished. Plasma renin activity increased during the IVT glycerol infusion and during water deprivation, but was largely unaffected by IVT glucose. Both IVT glycerol and glucose decreased renal sodium excretion. The possibility is discussed that the pronounced ability of IVT glycerol to depress the vasopressin release and thirst is not only due to dilution induced reduction of CSF [Na+], but also to an influence of glycerol on choroidal and/or transependymal Na+-transporting mechanisms.     

Biphasic change in plasma potassium concentration by mannitol infusion in rats.

Changes in plasma Na+([Na+]pl) and K+([K+]pl) concentrations were continuously measured in nephrectomized rats consequent to intravenous infusion of isosmotic mannitol (M group) or sucrose (S group) at 1.6 ml/100 g body wt. for 10 min, and for the following 30 min. The effect of a Ca2+ channel blocker (diltiazem, 0.1 mg/100 g body wt.) was also evaluated. In the S group, [K+]pl decreased during the infusion and rose above the control level after the discontinuance of infusion. In the M group, [K+]pl gradually increased even during the infusion and reached the same level as the S group at the end of the experiment. In both the S and M groups, [Na+]pl decreased, while PaCO2, pH, and HCO3- did not change. The increase in [K+]pl in the M group had two components. The first was a transient component as presented by the difference of [K+]pl between the M and S group. It might be attributable to K+ release from the intracellular space during a regulatory volume decrease consequent to cell swelling induced by isosmotic mannitol infusion. The second component of the increase in [K+]pl after the infusion was observed in both groups, and this increase might be related to the dilution of ions induced by the large amount of infused nonelectrolyte solution. Diltiazem suppressed both of these changes in [K+]pl, suggesting that they are related to Ca2+ channels. The results suggest that increase in [K]pl by isosmotic mannitol infusion is related to the effects of regulatory volume decrease and dilutional effect.     

Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells

The present study was performed to show the molecular identity of functionally characterized Mg transport pathways in rumen epithelial cells (REC) and to investigate the effects of extracellular [Mg] changes on their expression and activity. By using RT- PCR, Western blot, flow cytometry and immunocytochemistry, TRPM7, MagT1 and a Na+/Mg2+ exchanger were found in REC. Compared with control conditions ([Mg]e = 1.2 mM), a decreased or increased MagT1 (-30%; 20%) and Na+/Mg2+ exchanger (-25%; 40%) protein abundance was observed after a 24-h incubation of REC in low (0.12 mM)- and high (5 mM)-Mg medium, respectively. To determine the Mg transport capacity, [Mg2+]i changes were measured by use of mag-fura 2. The basal [Mg2]i (0.43 +/- 0.03 mM) was not influenced by the [Mg] of the pre-incubation medium. However, compared to control cells, REC incubated in low- or high-Mg medium showed significantly reduced (59%) and elevated (57%) Mg extrusion rates, respectively. In addition, they were characterized by an increased influx capacity (30-40%). In low-Mg cells the latter results mainly from a strong TRPM7 related transport component whereas in high-Mg cells the imipramine-sensitive, the Na+/Mg2+ exchanger-mediated transport component causes this effect. In conclusion, TRPM7, MagT1 and a Na+/Mg2+ exchanger are shown to be the main Mg transport proteins in REC and their expression and functional activity is influenced by the cellular Mg status. The latter responses permit adaptation of epithelial Mg absorption and enable REC to maintain a physiological [Mg2+]i which is a prerequisite for various cell functions.     

Acute reduction of extracellular sodium differentially affects receptor-mediated and K+-induced calcium uptake in PC12 pheochromocytoma cells

Using a rapid-quench method to measure 45Ca2+ uptake into PC12 cells in suspension, we have studied basal, carbachol-stimulated and K+-induced Ca2+ uptake under control conditions [( Na+]o = 130 mM) and in the presence of acutely lowered extracellular sodium concentration [( Na+]o = 65 mM). Acute reduction of [Na+]o stimulates basal and K+-evoked Ca2+ uptake, but reduces net carbachol-stimulated uptake. Since total Ca2+ uptake measured in the presence of carbachol under control and low [Na+]o conditions is unchanged, the reduction in carbachol-stimulated uptake is due to the increase in basal uptake induced by low [Na+]o. These results reconcile apparently conflicting data regarding a specific Na+ requirement for nicotinic acetylcholine receptor-mediated responses in PC12 cells and adrenal chromaffin cells and suggest a mechanism for loss of nicotinic acetylcholine receptor (nAChR) responsiveness to agonists under low Na+ conditions.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.