Activation of Na+,H+ exchanger produces vasoconstriction of renal resistance vessels

To evaluate the influence of the sodium/proton exchanger (Na⁺,H⁺ exchanger) on the constriction of rat resistance vessels and on the iliac artery, the isometric vasoconstrictions of renal resistance vessels and strips from iliac artery derived from Wistar-Kyoto rats were measured using a vessel myograph. The Na⁺,H⁺ exchanger was activated by intracellular acidification using propionic acid. Cytosolic pH (pH_i) and cytosolic free sodium concentration ([Na⁺]_i) in vascular smooth muscle cells were measured using the fluorescent dye technique. The activation of the Na⁺,H⁺ exchanger increased the [Na⁺]_i by 12.4 ± 1.3 mmol/L (n = 8). The activation of the Na⁺,H⁺ exchanger caused a contractile response of the renal resistance vessels (increase of tension, 1.5 ± 0.1 × 10⁻³ N; n = 13) and of the rat iliac artery (increase of tension, 7.5 ± 0.8 × 10⁻³ N; n = 5). The contractile response after activation of the Na⁺,H⁺ exchanger was significantly inhibited in the absence of external sodium or in the presence of amiloride, confirming the involvement of the Na⁺,H⁺ exchanger. The contractile response after activation of the Na⁺,H⁺ exchanger was significantly reduced in the absence of external calcium, after inhibition of calcium channels by nifedipine, and in the presence of an intracellular calcium antagonist 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the activation of the Na⁺,H⁺ exchanger consecutively caused transplasma membrane calcium influx. On the other hand, the inhibition of the Na⁺,Ca²⁺ exchanger by NiCl₂ significantly increased the vasoconstriction of renal resistance vessels after activation of the Na⁺,H⁺ exchanger. The activation of the Na⁺,H⁺ exchanger produces vasoconstriction by an increased cytosolic sodium concentration, inhibition of the Na⁺,Ca²⁺ exchanger, and activation of transplasma membrane calcium influx through potential dependent calcium channels.     

Myocardial recovery during post-ischemic reperfusion: Optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution

The effects of Na⁺ and Ca²⁺ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas’ solution. The concentration of Na⁺ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca²⁺, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110 mM/l Na⁺ and 1.2–1.8 mM/l Ca²⁺ in the reperfusate. A significantly greater postischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca²⁺ overload via Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.     

The Links Between Cellular Ca2+ and Na+/H+ Exchange in the Pathophysiology of Essential Hypertension

This review focuses on the mechanisms whereby the cytosolic Ca²⁺ regulates the ubiquitous Na⁺/H⁺ exchanger (NHE-1) and how these regulatory processes might modify the behavior of NHE-1 in essential hypertension. The pH setpoint for activation of the Na⁺/H⁺ exchanger is controlled by two interrelated and Ca²⁺-dependent pathways, namely, the protein kinase/phosphatase cascade and Ca²⁺/calmodulin. The cytoplasmic domain of NHE-1 contains elements responsive to serine/theorine kinases and a high affinity binding site to Ca²⁺/calmodulin. Phosphorylation of NHE-1 or the binding of the Ca²⁺/calmodulin complex to its binding site promotes an alkaline shift in the pH setpoint for the exchanger. It is suggested that, in essential hypertension, an increased cellular Ca²⁺ load or an enhanced external Ca²⁺ entry stimulate the NHE-1 through protein kinase/phosphatase and Ca²⁺/calmodulin systems, thereby increasing its activity. Am J Hypertens 1996;9:703–707     

Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

Ultrastructural study of calcium shift in ischemic/reperfused rat heart under treatment with dimethylthiourea, diltiazem and amiloride

Among factors underlying reperfusion injury are oxygen free radicals and Ca²⁺ influx via gated calcium channel or via Na⁺/H⁺–Na⁺/Ca²⁺ exchange which lead to calcium overload. The aim of the study was to ultrastructurally visualize the distribution of Ca²⁺ and to compare binding of calcium by the sarcolemma and calcium accumulation in mitochondria under therapy with an ·OH scavenger, dimethylthiourea (DMTU), Na⁺/H⁺ exchange inhibitor, amiloride, and calcium channel blocker, diltiazem, given alone or in combination to ischemic/reperfused hearts. Isolated working hearts subjected to 30 min ischemia and 30 min reperfusion were perfused with drugs added to the perfusate 15 min before ischemia and administered for the rest of the perfusion period. The cytochemical phosphate pyroantimonate method for localization of Ca²⁺ was used, and calcium distribution was analyzed with a computer image analyzer. All drugs given alone improved sarcolemmal ability to bind calcium. The best results were obtained with amiloride. All of the combined therapies gave even better results, but calcium accumulation in mitochondria diminished only with diltiazem therapy given alone or in combination with DMTU. Since the presence of Ca²⁺ deposits on the sarcolemma is believed to represent its normal function, and calcium sequestration by mitochondria reflects an increase in cytosolic calcium load, the lack of correlation between sarcolemmal and mitochondrial Ca²⁺ distribution might suggest impaired mechanisms of lowering cytoplasmic calcium or the existence of some mechanism other than Na⁺/Ca²⁺ exchange, mediated by activated Na⁺/H⁺ exchange. Received: 3 March 1997, Returned for 1. revision: 21 September 1997, 1. Revision received: 31 October 1997, Returned for 2. revision: 29 November 1997, 2. Revision received: 9 February 1998, Returned for 3. revision: 16 February 1998, 3. Revision received: 2 March 1998, Accepted: 3 March 1998     

Angiotensin II type 2 receptor inhibits expression and function of insulin receptor in rat renal proximal tubule cells

Both renin–angiotensin systems and insulin participate in kidney-involved blood pressure regulation. Activation of angiotensin II type 2 receptor (AT₂R) decreases sodium reabsorption in renal proximal tubule (RPT) cells, whereas insulin produces the opposite effect. We presume that AT₂R has an inhibitory effect on insulin receptor expression in RPT cells, which may affect renal sodium transport and therefore be of physiological or pathological significance. Our present study found that activation of AT₂R inhibited insulin receptor expression in a concentration and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. In the presence of a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19–31, 10^?6 mol/L) or a phosphatidylinositol 3 kinase inhibitor (wortmannin, 10^?6 mol/L), the inhibitory effect of AT₂R on insulin receptor was blocked, indicating that both PKC and phosphatidylinositol 3 kinase were involved in the signaling pathway. There was a linkage between AT₂R and insulin receptor which was determined by both laser confocal microscopy and coimmunoprecipitation. However, the effect of AT₂R activation on insulin receptor expression was different in RPT cells from spontaneously hypertensive rats (SHRs). Being contrary to the effect in WKY RPT cells, AT₂R stimulation increased insulin receptor in SHR RPT cells. Insulin (10^?7 mol/L, 15 minutes) enhanced Na⁺-K⁺-ATPase activity in both WKY and SHR RPT cells. Pretreatment with CGP42112 decreased the stimulatory effect of insulin on Na⁺-K⁺-ATPase activity in WKY RPT cells, whereas pretreatment with CGP42112 increased it in SHR RPT cells. It is suggested that activation of AT₂R inhibits insulin receptor expression and function in RPT cells. The lost inhibitory effect of AT₂R on insulin receptor expression may contribute to the pathophysiology of hypertension.     

PKC mediates GnRH activation of a Na/H exchanger in goldfish somatotropes

Previous results suggest that gonadotropin-releasing hormone (GnRH) stimulation of somatotropin secretion in goldfish involves activation of Na⁺/H⁺ exchange (NHE). We tested the hypothesis that GnRH alkalinizes intracellular pH (pH_i) via protein kinase C (PKC) activation of NHE. Two types of alkalinization responses were observed in identified goldfish somatotropes preloaded with the pH-sensitive dye BCECF; the rate of pH_i changes went from a neutral or slightly negative slope to either a positive or a less negative slope relative to control. Two GnRHs, the PKC-activating TPA, and dioctanoyl glycerol each caused an alkalinization in 70-90% of somatotropes. The PKC inhibitors, Bis II and Gö6976, the NHE inhibitor amiloride, or Na⁺-free solution attenuated TPA and GnRHs actions, suggesting that PKC mediates GnRH activation of NHE. Since amiloride and Na⁺-free solution caused acidification in somatotropes at rest, regulation of basal pH_i in these cells likely involves Na⁺ flux through amiloride-sensitive NHE.     

A functional interaction between TRPC/NCKX induced by DAG plays a role in determining calcium influx independently from PKC activation

Ca²⁺influx might occur through K⁺-dependent Na⁺/Ca²⁺ exchanger operating in reverse mode (rNCKX). In a cellular model different from platelets, an interaction between canonical transient receptor potential cation (TRPC) channels and NCX has been found. The aim of this study was to verify whether the TRPC/NCKX interaction operates in human platelets. Our results showed that the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced rNCKX-mediated Ca²⁺ influx through TRPC-mediated Na⁺ influx. DAG-induced activation of TRPC/NCKX occurs independently of protein kinase C (PKC) activation, as PKC inhibitor did not modify OAG-mediated Ca²⁺ influx. Moreover, as both rNCKX and TRPC inhibitors reduced OAG-induced platelet aggregation which, conversely, was increased by flufenamic acid, known to develop TRPC activity, it could be suggested that the TRPC/NCKX interaction has a role in OAG-dependent platelet aggregation.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

Can dapagliflozin have a protective effect against COVID-19 infection? A hypothesis

It has been reported that frequent occurrence of COVID-19 infection in these patients is associated with low cytosolic pH. During virus infection, serum lactate dehydrogenase (LDH) level excessively rises. LDH is a cytosolic enzyme and the serum level increases as the cell break down. When anaerobic conditions develop, lactate formation increases from pyruvate. Cell pH is regulated by very complex mechanisms. When lactate increases in the extracellular area, this symporter carries lactate and H⁺ ion into the cell, and the intracellular pH quickly becomes acidic. Paradoxically, Na⁺/H⁺ exchanger activation takes place. While H⁺ ion is thrown out of the cell, Na⁺ and Ca⁺² enter the cell. When Na⁺ and Ca⁺² increase in the cell, the cells swell and die. Dapagliflozin is a sodium-glucose cotransporter-2 inhibitor. Dapagliflozin has been reported to reduce lactate levels by various mechanisms. Also, it reduces oxygen consumption in tissues and causes the use of glucose in the aerobic pathway, thereby reducing lactate production. A lactate decrease in the environment reduces the activation of lactate/H⁺ symporter. Thus, the H ion pumping into the cell by this symporter is reduced and the cytosolic pH is maintained. Dapagliflozin also directly inhibits NHE. Thus, Na⁺ and Ca⁺² flow to the cell are inhibited. Dapagliflozin provides the continuation of the structure and functions of the cells. Dapagliflozin can prevent the severe course of COVID-19 infection by preventing the lowering of cytosolic pH and reducing the viral load.     

Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes

Phorbol myristate acetate (PM A) increased the formation of |³²P | PI 4,5-P, in ³²P-prelabeled human platelet. In saponin-permeabilized platelets, in which ³²P from exogenous |γ-³²P| ATP was incorporated into PI 4-P and PI 4,5-P₂, addition of 10 nM PMA resulted in increased formation of |³²P|PI 4,5-P₂ and |³²P|PI 4-P. In order to distinguish whether increased [³²P]PI 4,5-P₂ formation by PMA reflected merely an increase of [³²P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC.

However, 100 nM PMA did not stimulate [³²P]PI 4,s-P₂ formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P₂ inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca²⁺ -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca²'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca²⁺ -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p₂ and Ca²⁺ -independent PKC(nPKC).     

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca²⁺ imaging and two-photon Na⁺ imaging. GABA evoked Ca²⁺ transients in astrocytes that persisted in the presence of GABA_A and GABA_B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca²⁺ blocked GABA-induced Ca²⁺ transients, and depletion of Ca²⁺ stores with cyclopiazonic acid reduced Ca²⁺ transients by approximately 90%. This indicates that the Ca²⁺ transients depend on external Ca²⁺, but are mainly mediated by intracellular Ca²⁺ release, conforming with Ca²⁺-induced Ca²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca²⁺ transients, whereas the InsP₃ receptor blocker 2-APB inhibited the Ca²⁺ transients. GABA also induced Na⁺ increases in astrocytes, potentially reducing Na⁺/Ca²⁺ exchange. To test whether reduction of Na⁺/Ca²⁺ exchange induces Ca²⁺ signaling, we inhibited Na⁺/Ca²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca²⁺ transients in astrocytes. The significance of GABAergic Ca²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na⁺ rises that reduce Na⁺/Ca²⁺ exchange, thereby leading to a Ca²⁺ increase sufficient to trigger Ca²⁺-induced Ca²⁺ release via InsP₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Calcium and sodium control in hypoxic-reoxygenated cardiomyocytes

Summary When oxygen-deprived cardiomyocytes become energy depleted, they accumulate Na⁺ and Ca²⁺ in the cytosol. Influx of Ca²⁺ via the Na⁺/Ca²⁺ exchange mechanism seems to contribute to the development of Ca²⁺ overload, but Ca²⁺ overload may eventually also occur when this route is blocked. Hypoxic-reoxygenated cardiomyocytes in a state of severe overload of Na⁺ and Ca²⁺ can rapidly re-establish a normal cation control when oxidative energy production is re-initiated. The recovery of cellular Ca²⁺ control may be devided into three stages: first, sequestration of large amounts of Ca²⁺ into the sarcoplasmic reticulum; second, oscillatory movement of Ca²⁺ from and back into the sarcoplasmic reticulum and gradual extrusion across the sarcolemma; third, re-establishment of constant low cytosolic Ca²⁺ concentrations. When the Na⁺/Ca²⁺ exchanger is inhibited, extrusion of Ca²⁺ from the cells' interior is impaired and oscillatory Ca²⁺ movements between cytosol and sarcoplasmic reticulum continue for long time. Thus, the functions of the sarcoplasmic reticulum and the Na⁺/Ca²⁺ exchanger are of crucial importance for the recovery of Ca²⁺ control in reoxygenated cardiomyocytes. In re-energized cardiomyocytes, a persistent elevation of the cytosolic Ca²⁺ concentration provokes maximal force development and consecutive mechanical cell injury (oxygen paradox). This injury can be prevented when the contractile machinery is inhibited during the initial phase of reoxygenation as long as necessary for the re-establishment of a normal cytosolic Ca²⁺ control.     

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH_i-regulating transport systems involved is the Na⁺/H⁺ exchanger. Inhibitors of the Na⁺/H⁺ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na⁺ and secondary Ca²⁺ influx. Under reperfusion conditions Na⁺/H⁺ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na⁺/H⁺ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.     

Impact of conditioning hyperglycemic on myocardial infarction rats:Cardiac cell survival factors

While clinical data have suggested that the diabetic heart is more susceptible to ischemic heart disease(IHD),animal data have so far pointed to a lower probability of IHD. Thus,the aim of this present review is to look at these conflicting results and discuss the protective mechanisms that conditioned hyperglycemia may confer to the heart against ischemic injury. Several mechanisms have been proposed to explain the cardioprotective action of high glucose exposure,namely,upregulation of anti-apoptotic factor Bcl-2,inactivation of pro-apoptotic factor bad,and activation of pro-survival factors such as protein kinase B(Akt),vascular endothelial growth factor(VEGF),hypoxia inducible factor-1α and protein kinase C-ε. Indeed,cytosolic increase in Ca2+ concentration,the mitochondrial permeability transition pore,plays a key role in the genesis of ischemic injury. Previous studies have shown that the diabetic heart decreased Na+/Ca2+ and Na+/H+ exchanger activity and as such it accumulates less Ca2+ in cardiomyocyte,thus preventing cardiac injury and the associated heart dysfunctions. In addition,the expression of VEGFin diabetic animals leads to increased capillary density before myocardial infarction. Despite poor prognostic in the long-term,all these results suggest that diabetes mellitus and consequently hyperglycemia may indeed play a cardioprotective role against myocardial infarction in the short term.     

Cytosolic sodium concentration regulates contractility of cardiac muscle

Summary The present chapter provides experimental evidence to show that intracellular Na⁺ concentration regulates cardiac contractility effectively by altering intracellular Ca²⁺ concentration via the Na-Ca exchange. This steep coupling between the Na⁺ and Ca²⁺ electrochemical gradients implies that a change in intracellular Na⁺ concentration is accompanied by a concomitant change in intracellular Ca²⁺ concentration (and, therefore, contractility). Under the physiologic conditions, each cardiac action potential alters intracellular Na⁺ concentration in a dynamic manner. Therefore, Na-Ca exchange can regulate cardiac contraction from a beat-to-beat basis.     

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Ca²⁺ signaling regulates cell function. This is subject to modulation by H⁺ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) or [H⁺] ([H⁺]_i) can become compartmentalized, leading potentially to complex spatial Ca²⁺/H⁺ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H⁺]_i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca²⁺]_i rise, independent of sarcolemmal Ca²⁺ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H⁺ uncaging from 2-nitrobenzaldehyde also raised [Ca²⁺]_i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H⁺ uncaging into buffer mixtures in vitro demonstrated that Ca²⁺ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H⁺-evoked [Ca²⁺]_i rise. Raising [H⁺]_i tonically at one end of a myocyte evoked a local [Ca²⁺]_i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca²⁺ transport into the acidic zone via Ca²⁺/H⁺ exchange on diffusible HDPs and ATP molecules, energized by the [H⁺]_i gradient. Ca²⁺ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg²⁺ overload or by ATP depletion, maneuvers that reduce the Ca²⁺-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca²⁺/H⁺ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca²⁺/H⁺ coupling is likely to be of general importance in cell signaling.Most cells are exquisitely responsive to calcium (Ca²⁺) (1) and hydrogen (H⁺) ions (i.e., pH) (2). In cardiac myocytes, Ca²⁺ ions trigger contraction and control growth and development (3), whereas H⁺ ions, which are generated or consumed metabolically, are potent modulators of essentially all biological processes (4). By acting on Ca²⁺-handling proteins directly or via other molecules, H⁺ ions exert both inhibitory and excitatory effects on Ca²⁺ signaling. For example, in the ventricular myocyte, H⁺ ions can reduce Ca²⁺ release from sarcoplasmic reticulum (SR) stores, through inhibition of the SR Ca²⁺ ATPase (SERCA) pump and ryanodine receptor (RyR) Ca²⁺ channels (5, 6). In contrast, H⁺ ions can enhance SR Ca²⁺ release by stimulating sarcolemmal Na⁺/H⁺ exchange (NHE), which raises intracellular [Na⁺] and reduces the driving force for Ca²⁺ extrusion on Na⁺/Ca²⁺ exchange (NCX), leading to cellular retention of Ca²⁺ (7, 8). Ca²⁺ signaling is thus subservient to pH.Cytoplasmic Ca²⁺ and H⁺ ions bind avidly to buffer molecules, such that <1% of all Ca²⁺ ions and <0.001% of all H⁺ ions are free. Some of these buffers bind H⁺ and Ca²⁺ ions competitively, and this has been proposed to be one mechanism underlying cytoplasmic Ca²⁺/H⁺ coupling (9). Reversible binding to buffers greatly reduces the effective mobility of Ca²⁺ and H⁺ ions in cytoplasm (10, 11) and can allow for highly compartmentalized ionic microdomains, and hence a spatially heterogeneous regulation of cell function. In cardiac myocytes under resting (diastolic) conditions, the cytoplasm-averaged concentration of free [Ca²⁺] ([Ca²⁺]_i) and [H⁺] ([H⁺]_i) ions is kept near 10⁻⁷ M by membrane transporter proteins. Thus, [H⁺]_i is regulated by the balance of flux among acid-extruding and acid-loading transporter proteins at the sarcolemma [e.g., NHE and Cl⁻/OH⁻ (CHE) exchangers, respectively] (4). Similarly, the activity of SERCA and NCX proteins returns [Ca²⁺]_i to its diastolic level after evoked signaling events (3, 12). Despite these regulatory mechanisms, cytoplasmic gradients of [H⁺]_i and [Ca²⁺]_i do occur in myocytes and are an important part of their physiology. Gradients arise from local differences in transmembrane fluxes that alter [H⁺]_i or [Ca²⁺]_i. For example, spatial [H⁺]_i gradients are produced when NHE transporters, expressed mainly at the intercalated disk region, are activated (4, 13) or when membrane-permeant weak acids, such as CO₂, are presented locally (14). Similarly, release of Ca²⁺ through a cluster of RyR channels in the SR produces [Ca²⁺]_i nonuniformity in the form of Ca²⁺ sparks (15). Given the propensity of cytoplasm to develop ionic gradients, it is important to understand their underlying mechanism and functional role.The present work demonstrates a distinct form of spatial interaction between Ca²⁺ and H⁺ ions. We show that cytoplasmic [H⁺] gradients can produce stable [Ca²⁺]_i gradients, and vice versa, and that this interaction is mediated by low-molecular-weight (mobile) buffers with affinity for both ions. We demonstrate that the diffusive counterflux of H⁺ and Ca²⁺ bound to these buffers comprises a cytoplasmic Ca²⁺/H⁺ exchanger. This acts like a “pump” without a membrane, which can, for instance, recruit Ca²⁺ to acidic cellular microdomains. Cytoplasmic Ca²⁺/H⁺ exchange adds a spatial paradigm to our understanding of Ca²⁺ and H⁺ ion signaling.     

Intracellular Levels of Na+ and TTX-sensitive Na+ Channel Current in Diabetic Rat Ventricular Cardiomyocytes

Intracellular Na⁺ ([Na⁺] _i ) is an important modulator of excitation–contraction coupling via regulating Ca²⁺ efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na⁺] _i in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na⁺ channel current (I _Na) density. We recorded either [Na⁺] _i or [Ca²⁺] _i by using a fluorescent Na⁺ indicator (SBFI-AM or Na-Green) or a Ca²⁺ indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na⁺] _i and [Ca²⁺] _i at rest, and also [Na⁺] _i during pacing with electrical field stimulation in a range of 0.2–2.0 Hz stimulation frequency. In order to test the possible contribution of Na⁺/H⁺ exchanger (NHE) to [Na⁺] _i , we examined the free cytoplasmic [H⁺] _i changes from time course of [H⁺] _i recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na⁺] _i in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na⁺] _i was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca²⁺] _i in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH _i in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na⁺ loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H⁺-flux. NHE protein level remained unchanged, while protein levels of Na⁺/K⁺ ATPase and Na⁺/Ca²⁺ exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I _Na plays an important role in altered electrical activity with less Na⁺ influx during contraction, and an increased [Ca²⁺] _i load in these cells seems to be independent of [Na⁺] _i . The data with insulin treatment suggest further a recent balance between Na⁺ influx and efflux proteins associated with the [Na⁺] _i , particularly during diabetes.     

Diabetes-induced activation of protein kinase C inhibits store-operated Ca<Superscript>2+</Superscript> uptake in rat retinal microvascular smooth muscle

Aims/Hypothesis To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca²⁺ influx pathways in retinal microvascular smooth muscle cells.Methods Cytosolic Ca²⁺ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca²⁺ influx was tested by measuring rises in [Ca²⁺]_i with KCl (100 mmol/l) and store-operated Ca²⁺ influx was assessed by depleting [Ca²⁺]_i stores with Ca²⁺ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca²⁺ on adding 2 mmol/l or 10 mmol/l Ca²⁺solution.Results Ca²⁺ entry through voltage-dependent L-type Ca²⁺ channels was unaffected by diabetes. In contrast, store-operated Ca²⁺ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca²⁺ influx. Diabetic rats injected daily with insulin had store-operated Ca²⁺ influx rates similar to non-diabetic control rats. The reduced Ca²⁺ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKC antagonist LY379196 (100 nmol/l) also reversed the poor Ca²⁺ influx although its action was less efficacious than staurosporine.Conclusion/interpretation These results show that store-operated Ca²⁺ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKC seems to play a role in mediating this effect.Abbreviations DAG Diacylglycerol - PKC protein kinase C - [Ca²⁺]_i intracellular calcium concentration - STZ streptozotocin - SPP staurosporine - SR sarcoplasmic reticulum - MVSM microvascular smooth muscle - CPA cyclopiazonic acid - PMA phorbol myristate acetate - VDCC voltage-dependent Ca²⁺ channels