Hypomagnesemia: renal magnesium handling.

Magnesium is an important constituent of the intracellular space that affects a number of intracellular and whole body functions. Magnesium balance depends on intake and renal excretion, which is regulated mainly in the thick ascending limb of the loop of Henle. The complex hormonal modulation that responds to changes in plasma concentration of other ions such as calcium and potassium is lacking for magnesium. As a result, negative magnesium balance results in a prompt decrease in plasma magnesium concentration, and hypermagnesemia accompanies renal failure with magnesium accumulation. Hypomagnesemia may result from gastrointestinal losses or renal losses, the latter due to primary renal magnesium wasting or in association with sodium loss. Hypomagnesemia may arise together with and contribute to the persistence of hypokalemia and hypocalcemia. The major direct toxicity of hypomagnesemia is cardiovascular. When urgent correction of hypomagnesemia is required, as with myocardial ischemia, post cardiopulmonary bypass, and torsades de pointes, intravenous or intramuscular magnesium sulfate should be used. Oral magnesium preparations are available for chronic use.

Effects of intracellular magnesium on calcium, potassium and chloride channels.

Intracellular free magnesium activity approximates 0.6-0.8 mM. Variations in concentration around this physiologic range profoundly affect current flow through calcium, potassium and chloride channels. These effects are exerted on both inwardly and outwardly directed currents and appear to be independent of the state of phosphorylation of the channel. In the heart, these effects are manifested in changes in action potential duration, which may help to explain postulated links between hypomagnesemia and arrhythmias. In the kidney, effects of magnesium on ion channels may subserve a protective role and may explain how potassium balance is altered in magnesium depletion syndromes.

Cytosolic magnesium modulates calcium channel activity in mammalian ventricular cells.

The effect of cytosolic free Mg2+ concentration on the regulation of myocardial function was studied by dialyzing isolated guinea pig ventricular myocytes with different internal Mg2+ concentrations [( Mg2+]i). We found that elevation of [Mg2+]i shortened the action potential and suppressed the Ca2+ current. Mean values recorded for action potential duration in cells dialyzed with solutions containing 0, 1.3, and 9.4 mM Mg2+ were 620 +/- 40, 400 +/- 25, and 60 +/- 10, respectively. The suppressive effect of [Mg2+]i on the action potential duration correlated significantly with the suppressive effects of [Mg2+]i on the Ca2+ current. In cells dialyzed with nominally zero Mg2+, calcium current was prominent (3.5 +/- 0.58 nA). At [Mg2+]i of 1.4 mM, calcium current was significantly smaller than in zero [Mg2+]i and was almost completely inhibited by dialysis of the cell with 9.4 mM Mg2+. The Mg2+-induced block of the Ca2+ current was due to steady-state inactivation of the high threshold calcium channel. The block was observed in the presence or absence of adenosine 3',5'-cylic monophosphate and was not reversed by elevation of external Ca2+ concentration, addition of adrenaline, or large negative potentials. These data suggest that cytosolic Mg2+ regulates Ca2+ channel activity by a novel mechanism, unrelated to its effect as a blocking particle of the open channel.

Requirement for extracellular calcium or magnesium in mitogen-induced activation of human peripheral blood lymphocytes.

The importance of calcium in lymphocyte activation is well recognized, but the levels of extracellular ionized free calcium (Ca++) necessary for lymphocyte proliferation via various pathways have not been investigated in detail. We studied the ability of a lectin mitogen (PHA) and a calcium ionophore (ionomycin) to induce interleukin 2 receptors, interleukin 2 (IL2) production, and proliferation over various concentrations of extracellular Ca++. Reducing the Ca++ levels from the normal 200 microM to 10 microM in PHA-stimulated cultures partially inhibited IL2 receptor expression, IL2 production, and subsequent proliferation. At 1 microM Ca++, both IL2 activity and proliferation were eliminated, but partial IL2 receptor expression was still observed. Ionomycin did not induce any of these events in cultures where the extracellular Ca++ concentration was below 100 microM. Restoring calcium in the medium resulted in normal levels of IL2 receptor expression, IL2 activity, and proliferation when PBL were stimulated with either mitogen. Exogenous magnesium partially restored these events in PHA-stimulated cultures, but had no effect when ionomycin was used as the mitogen. These data indicate that stimulation by ionomycin is much more dependent upon the levels of extracellular Ca++ than is PHA. Extracellular calcium also appears to be necessary subsequent to IL2 receptor acquisition, since the latter was seen without IL2 activity or proliferation at very low extracellular Ca++, and IL2 failed to restore the proliferative response under these conditions. The data also suggest that PHA, but not ionomycin, can activate lymphocytes via a magnesium-dependent pathway, or that PHA has a lower specificity for divalent cation cofactors.

Benzodiazepines stimulate sodium ion transport in frog skin epithelium.

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.

Thiazides stimulate calcium absorption in urinary bladder of winter flounder.

Thiazides inhibit voltage-independent NaCl absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutral mucosal Na/Cl co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCl absorption, we studied the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and intracellular electrical potential in short-circuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10(-4) M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10(-4) M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyperpolarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCl absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule.

Relationships between calcium and chloride transport in frog skin glands.

Frog skin gland, a furosemide-sensitive Cl(-)-secreting epithelium, exhibits Cl(-)-dependent Ca2+ secretion in response to stimulation by beta-adrenergic agonists. In this study, we further explored the relationships between Cl- and Ca2+ secretion in frog skin using 45Ca fluxes and short-circulating technique. On addition of isoproterenol (ISO) or 8-(p-chlorophenylthio)-cAMP, a significant positive correlation was demonstrated between Ca2+ secretion and Cl- secretion. Because Cl- transport in other Cl(-)-transporting epithelia may be modulated by prostaglandins or by changes in cytosolic Ca2+ activity, in addition to modulation by cAMP, we also examined the effects of prostaglandins (PG)E2 and F2 alpha, indomethacin (INDO), and the calcium ionophore A23187. Treatment with PGE2, PGF2 alpha, or A23187 at a dose of 10(-5) M resulted in marked stimulation in the amiloride-resistant short-circuit current, a reflection of Cl- secretion. This current was inhibited by furosemide addition or removal of Cl- from the bathing medium. However, and in contrast to stimulation with ISO or cAMP, PGE2, PGF2 alpha, and A23187 failed to induce Ca2+ secretion. In addition, the stimulation of Cl- secretion by A23187 was abolished by INDO (10(-6) M) pretreatment. Thus frog skin glands secrete Cl- via two mechanisms: one mediated by beta-adrenergic-cAMP stimulation and the other by activation of prostaglandin metabolism induced by changes in cytosolic Ca2+. Only the former pathway is associated with Ca2+ secretion. Furthermore, to account for the Cl- dependence of Ca2+ secretion, we postulate the existence of a Ca2+-Cl- cotransport system stimulated by cAMP.

Microelectrode study of intracellular pH in frog skin: dependence on serosal chloride.

Replacement of external chloride has been known to reduce Na+ transport across whole frog skin. However, the sidedness and mechanism of the phenomenon have been unclear. In the present study, transepithelial current (IT), transepithelial resistance (RT), and basolateral membrane potential measured both with reference micropipettes (psi sc) and pH-selective microelectrodes (EscH) were monitored in isolated epithelial sheets from frog skin; removal of the underlying dermis facilitates ionic exchange across the basolateral membranes. The intracellular hydronium ion activity (acH) was 58 +/- 4 nM (means +/- SE) when the extracellular hydronium activity was 25 +/- 1 nM under base-line conditions. This measurement is equivalent to an intracellular pH (pHc) of 7.24 +/- 0.03 at an extracellular pH of 7.60 +/- 0.01, in reasonable agreement with estimates obtained by 31P- and 19F-nuclear magnetic resonance (NMR) analyses of frog skin. Complete replacement of mucosal Cl- by gluconate had variable effects on tissue current and resistance from preparation to preparation. The same ionic substitution on the serosal side uniformly produced a prompt reversible decrease in IT, increase in RT, and a substantial membrane depolarization of the short-circuited skins. In most of the preparations, the depolarization was preceded by a small hyperpolarization of 0.5-3.5 mV. The replacement of serosal Cl- also produced a fall in intracellular hydronium ion activity of 33 +/- 10 nM. The present date are consistent with the concept that serosal replacement of Cl- alkalinizes the cells by either favoring HCO3- entry or blocking HCO3- exit through a Cl- HCO3 antiport at the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP- and beta-adrenergic-stimulated chloride-dependent Ca2+ secretion in frog skin.

This study examined the possible existence and nature of Ca2+ transport in frog skin using 45Ca fluxes and short-circuiting technique. Following the addition to full-thickness frog skin (FTFS) of 8-[p-chlorophenylthio]cAMP (8-CPT-cAMP), forskolin, or 1-methyl-3-isobutylxanthine, the secretory Ca2+ flux increased severalfold, inducing net Ca2+ secretion. The absorptive flux was unchanged. Isoproterenol (10(-6)M) reproduced the effects of cAMP on Ca2+ secretion (-3.76 +/- 0.80 nmol X cm-2 X h-1 vs. +0.04 +/- 0.05 in control) while vasopressin and parathyroid hormone did not alter Ca2+ fluxes. Because FTFS contains subepidermal glands capable of Cl- secretion in response to beta-adrenergic stimulation, split-thickness frog skin (STFS) consisting of the gland-free Na-absorbing surface epithelium was used to localize the anatomic site of Ca2+ secretion. In STFS, addition of 8-CPT-cAMP or isoproterenol failed to induce Ca2+ secretion, suggesting that this transport in FTFS is localized in skin glands. Additional studies explored the relationship between Ca2+ and Cl- transport in FTFS. Furosemide prevented the stimulation of both Ca2+ and Cl- secretion. Removal of Cl- from the bathing medium abolished Ca2+ secretion. Thus, FTFS exhibits a beta-adrenergic, cAMP-stimulated net Ca2+ secretion that is Cl- dependent. As this effect is not observed in STFS, the pathway of Ca2+ secretion in frog skin is probably localized in the subepidermal glandular epithelium in association with Cl- secretion. Frog skin glands may represent a useful model for the study of Ca2+ transport in Cl--transporting epithelia.

Intracellular calcium activity in split frog skin epithelium: effect of cAMP.

Measurement of intracellular calcium activity (acCa) by ion-selective microelectrodes has previously been technically limited to relatively large cells (greater than or equal to 20 micron). We now report results obtained with this technique in the small epithelial cells (less than or equal to 10 micron) of split frog skin using microelectrodes having an outer tip diameter of less than 0.2 micron. The basolateral membrane potential was measured with Ca2+-selective microelectrodes (EscCa) and with reference micropipettes (psi sc) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions, acCa was measured to be 215 +/- 39 nM (mean +/- SE), in close agreement with the mean values estimated from published data obtained with Necturus proximal tubule. Stimulation of Na+ transport across six skins with 1 mM serosal 8 p-chlorophenylthio-3,5' cyclic AMP (CPTcAMP) increased acCa by a factor of 2.6 +/- 0.6. The increase in acCa preceded the CPTcAMP-induced increase in Isc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca2+ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.

Effects of two models of hypercalcemia on renal acid base metabolism.

The effects of two models of chronic hypercalcemia on renal acid-base metabolism were studied in rats. In the first series of experiments, rats were rendered hyperparathyroid by the autologous grafting of 20 to 24 parathyroid glands into a single recipient. Hypercalcemia (5.48 +/- 0.03 mEq/liter in high PTH animals, 4.96 +/- 0.06 mEq/liter in pair-fed controls, P less than 0.001) occurred as did metabolic alkalosis (plasma total carbon dioxide 25.44 +/- 0.47 mEq/liter vs. 23.84 +/- 0.57 in controls, P less than 0.05). The rise in total carbon dioxide was in part a renal tubular effect since urine pH was lower (6.77 +/- 0.04 vs. 6.95 +/- 0.04, day 5, P less than 0.01) bicarbonaturia less (165 +/- 26 vs. 283 +/- 28 mumoles/24 hr, day 5, P less than 0.01) and titratable acid (TA) excretion increased (164 +/- 43.4 vs. 48.2 +/- 2.53 mEq/24 hr, day 5, P less than 0.01) in hyperparathyroid animals vs. pair-fed controls. To test the specific role of hypercalcemia versus PTH in this effect, normoparathyroid animals were treated with 1.25 (OH)2 vitamin D3 or SHAM injected, Urinary cAMP was reduced in these animals (0.030 +/- 0.004 mumoles/8 hr) compared to hyperparathyroid rats (0.055 +/- 0.01 mumoles/8 hr P less than 0.05) suggesting differences i PTH levels. Hypercalcemia occurred in 1,25(OH)2 vitamin D treated animals as did increased plasma total carbon dioxide and urinary TA while urinary bicarbonate excretion and urinary pH were reduced. Because hypercalcemia was associated with elevated total carbon dioxide in both models, it is proposed that chronic hypercalcemia stimulated renal acid excretion and in a sustained manner results in metabolic alkalosis, at least in part, on a renal basis.