Essential role of Kir5.1 channels in renal salt handling and blood pressure control.

Supplementing diets with high potassium helps reduce hypertension in humans. Inwardly rectifying K+ channels Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the basolateral membrane of distal renal tubules and contribute to Na+ reabsorption and K+ secretion through the direct control of transepithelial voltage. To define the importance of Kir5.1 in blood pressure control under conditions of salt-induced hypertension, we generated a Kcnj16 knockout in Dahl salt-sensitive (SS) rats (SSKcnj16-/-). SSKcnj16-/- rats exhibited hypokalemia and reduced blood pressure, and when fed a high-salt diet (4% NaCl), experienced 100% mortality within a few days triggered by salt wasting and severe hypokalemia. Electrophysiological recordings of basolateral K+ channels in the collecting ducts isolated from SSKcnj16-/- rats revealed activity of only homomeric Kir4.1 channels. Kir4.1 expression was upregulated in SSKcnj16-/- rats, but the protein was predominantly localized in the cytosol in SSKcnj16-/- rats. Benzamil, but not hydrochlorothiazide or furosemide, rescued this phenotype from mortality on a high-salt diet. Supplementation of high-salt diet with increased potassium (2% KCl) prevented mortality in SSKcnj16-/- rats and prevented or mitigated hypertension in SSKcnj16-/- or control SS rats, respectively. Our results demonstrate that Kir5.1 channels are key regulators of renal salt handling in SS hypertension.

Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells.

BACKGROUND/AIMS: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. METHODS: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. RESULTS: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. CONCLUSIONS: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

Cardiac sympathetic afferent stimulation induces salt-sensitive sympathoexcitation through hypothalamic epithelial Na+ channel activation.

The cardiac sympathetic afferent (CSA), which plays an important role in heart-brain communication for sympathoexcitation, is stimulated in heart failure. Additionally, high salt intake leads to further sympathoexcitation due to activation of hypothalamic epithelial Na(+) channels (ENaCs) in heart failure. In the present study, we stimulated the CSA in adult male mice by epicardial application of capsaicin and using ethanol as a control to determine whether CSA stimulation led to activation of hypothalamic ENaCs, resulting in salt-induced sympathoexcitation. Three days after capsaicin treatment, an upregulation of hypothalamic α-ENaCs, without activation of mineralocorticoid receptors, was observed. We also examined expression levels of the known ENaC activator TNF-α. Hypothalamic TNF-α increased in capsaicin-treated mice, whereas intracerebroventricular infusion of the TNF-α blocker etanercept prevented capsaicin-induced upregulation of α-ENaCs. To examine brain arterial pressure (AP) sensitivity toward Na(+), we performed an intracerebroventricular infusion of high Na(+)-containing (0.2 M) artificial cerebrospinal fluid. AP and heart rate were significantly increased in capsaicin-treated mice compared with control mice. CSA stimulation also caused excitatory responses with high salt intake. Compared with a regular salt diet, the high-salt diet augmented AP, heart rate, and 24-h urinary norepinephrine excretion, which is an indirect marker of sympathetic activity with mineralocorticoid receptor activation, in capsaicin-treated mice but not in ethanol-treated mice. Treatment with etanercept or the ENaC blocker benzamil prevented these salt-induced excitatory responses. In summary, we show that CSA stimulation leads to an upregulation of hypothalamic α-ENaCs mediated via an increase in TNF-α and results in increased salt sensitivity.

Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale.

BACKGROUND: The involvement of sodium-hydrogen exchangers (NHE) has been described in the pathophysiology of diseases including ischemic heart and brain diseases, cardiomyopathy, congestive heart failure, epilepsy, dementia, and neuropathic pain. Synthetic NHE inhibitors have not achieved much clinical success; therefore, plant-derived phytoconstituents may be explored as NHE inhibitors. METHODS: In the present study, the NHE inhibitory potential of hydroalcoholic and alkaloidal fractions of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale was evaluated. The different concentrations of hydroalcoholic and alkaloidal extracts of the selected plants were evaluated for their NHE inhibitory activity in the platelets using the optical swelling assay. RESULTS: Among the hydroalcoholic extracts, the highest NHE inhibitory activity was shown by M. domestica (IC50=2.350 ± 0.132 µg/mL) followed by Musa × paradisiaca (IC50=7.967 ± 0.451 µg/mL), D. carota (IC50=37.667 ± 2.517 µg/mL), and S. officinale (IC50=249.330 ± 1.155 µg/mL). Among the alkaloidal fractions, the highest NHE inhibitory activity was shown by the alkaloidal fraction of Musa × paradisiacal (IC50=0.010 ± 0.001 µg/mL) followed by D. carota (IC50=0.024 ± 0.002 µg/mL), M. domestica (IC50=0.031 ± 0.005 µg/mL), and S. officinale (IC50=4.233 ± 0.379 µg/mL). The IC50 of alkaloidal fractions was comparable to the IC50 of synthetic NHE inhibitor, EIPA [5-(N-ethyl-N-isopropyl)amiloride] (IC50=0.033 ± 0.004 µg/mL). CONCLUSIONS: It may be concluded that the alkaloidal fractions of these plants possess potent NHE inhibitory activity and may be exploited for their therapeutic potential in NHE activation-related pathological complications.

Hypericum perforatum differentially affects corticosteroid receptor-mRNA expression in human monocytic U-937 cells.

A dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis represents a prominent finding in major depression, possibly related to a dysfunction of the corticosteroid receptor system. Antidepressants are involved in the restoration of the altered feed-back mechanism of the HPA-axis, probably via normalization of corticosteroid receptor functions. Since Hypericum perforatum has antidepressive properties, we here examined its putative actions on glucocorticosteroid receptor mRNA levels in human blood cells as a peripheral model for neuroendocrine effects in human brain cells. Our data show that Hypericum (LI 160) affects the cellular mRNA levels of both, the glucocorticoid receptor (GR)-α and its inhibitory counterpart, the GR-ß, at clinically-relevant concentrations. Under these conditions, a bimodal effect was observed. Dose-response studies suggest a rather small effective concentration range and time-effect data show a primary and transient up-regulation of GR-α mRNA levels and a down-regulation of GR-ß mRNA levels after 16 h of treatment. The sodium channel blocker benzamil neutralized the effects of Hypericum, pointing to an at least partial mechanism of action via this pathway. In conclusion, Hypericum treatment differentially affects GR-mRNA levels in the human system. Our data suggest a bimodal effect on GR, resulting in a time-and dose-related modification of GR-mediated cellular effects. Such a mechanism has been alleged as an important way of action for a number of antidepressants. It is the first time that a specific effect on both receptors, especially on the subtype of GR-ß, is shown under antidepressive treatment in a human system under in vitro conditions.

Effects of central sodium on epithelial sodium channels in rat brain.

We evaluated the effects of intracerebroventricular (icv) infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial Na(+) channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11beta-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF Na(+) concentration ([Na(+)]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. Na(+)-rich aCSF increased beta-ENaC mRNA and immunoreactivity in the choroid plexus and increased alpha- and beta-ENaC immunoreactivities in the ependyma. Na(+)-rich aCSF increased alpha- and beta-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in beta-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, Na(+)-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [Na(+)] in the control, but not Na(+)-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [Na(+)] by Na(+)-rich aCSF. These results suggest that CSF Na(+) upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of Na(+) homeostasis in the brain.

Acute acidification or amiloride treatment suppresses the ability of Hsp70 to inhibit heat-induced apoptosis.

Inhibition of stress-induced apoptosis by the molecular chaperone protein Hsp70 is a contributing factor in tumorigenesis and suppression of this ability could increase the effectiveness of anti-tumor therapy. Tumor cells exist in an acidic environment and acute acidification can sensitize tumor cells to heat-induced cell death. However, the ability of Hsp70 to prevent apoptosis under these conditions has not been examined. The effect of acute acidification on heat-induced apoptosis was examined in a human T-cell line with tetracycline-regulated Hsp70 expression. Apoptosis was inhibited in cells exposed to hyperthermia in acidic media when examined 6 h after the heat stress, but resumed if cells were returned to physiological pH during this recovery period. Long-term proliferation assays showed that acute acidification sensitized cells to heat-induced apoptosis. Hsp70 expressing cells were also sensitized and this was correlated with a reduced ability to suppress the activation of JNK (c-jun N-terminal kinase), Bax and caspase-3. Further sensitization could be achieved with the NHE1 (Na(+)/H(+) exchanger) inhibitor HMA (5-(N, N-hexamethylene) amiloride), which potentiated JNK activation in heat-shocked cells. These results demonstrate that the ability of Hsp70 to suppress apoptosis is compromised when cells are exposed to hyperthermia in an acidic environment, which is correlated with an impaired ability to inhibit JNK activation.

Effects of bafilomycin A1, a vacuolar type H+ ATPase inhibitor, on the thermosensitivity of a human pancreatic cancer cell line.

PURPOSE: It has been known that the thermosensitivity of tumour cells can be increased by lowering intra-cellular pH (pHi) by inhibiting pHi control mechanisms. The pHi is partially controlled by transport of H+ from cytoplasm into endocytic and secretary systems in the cells mediated by vacuolar type H+ATPase and also by transport of H+ through plasma membrane. METHODS: This study investigated the effects the bafilomycine A1, an inhibitor of the vacuolar type H+ATPase and the EIPA, an inhibitor of the Na+/H+ exchanger in plasma membrane, on thermosensitivity of AsPC-1 cells, a human pancreatic cancer cell line. It also investigated the effects of combination of bafilomycine A1 and EIPA. RESULTS: The treatment of cancer cells with bafilomycine A1 or EIPA individually slightly lowered pHi of the cells in vitro and increased the thermosensitivity of the cells. CONCLUSION: The combination of these two drugs significantly lowered pHi and increased thermosensitivity of cancer cells in vitro and enhanced the heat-induced the growth delay of AsPC-1 tumours grown s.c in the legs of BALB/cA nude mice.

Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response.

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P < 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

On the interaction between amiloride and its putative alpha-subunit epithelial Na+ channel binding site.

The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are alphaSer-583, betaGly-525, and gammaGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the beta and gamma glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the alpha subunit resulted in moderate (<5-fold) weakening of amiloride K(i). We investigated the role of alphaSer-583 in amiloride binding by systematically mutating alphaSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a beta-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at alpha583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some alphaSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that alphaSer-583 may have a role in ion permeation through ENaC.

Heat-induced changes in intracellular Na+, pH and bioenergetic status in superfused RIF-1 tumour cells determined by 23Na and 31P magnetic resonance spectroscopy.

The acute effects of hyperthermia on intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) were investigated in superfused Radiation Induced Fibrosarcoma-1 (RIF-1) tumour cells using shift-reagent-aided 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy. Hyperthermia at 45 degrees C for 30 min produced a 50% increase in Na, a 0.42 unit decrease in pHi and a 40-45% decrease in NTP/P(i). During post-hyperthermia superfusion at 37 degrees C, pHi and NTP/P(i) recovered to the baseline value, but Na initially decreased and then increased to the hyperthermic level 60 min after heating. Hyperthermia at 42 degrees C caused only a 15-20% increase in Nai+. In the presence of 3 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, the increase in Nai+ during 45 degrees C hyperthermia was attenuated, suggesting that the heat-induced increase in Nai+ was mainly due to an increase in Na+/H+ anti-porter activity. EIPA did not prevent hyperthermia-induced acidification. This suggests that pHi is controlled by other ion exchange mechanisms in addition to the Na+/H+ exchanger. EIPA increased the thermo-sensitivity of the RIF-1 tumour cells only slightly as measured by cell viability and clonogenic assays. The hyperthermia-induced irreversible increase in Nai+ suggests that changes in transmembrane ion gradients play an important role in cell damage induced by hyperthermia.

Putative role of epithelial sodium channels (ENaC) in the afferent limb of cardio renal reflexes in rats.

Recent studies suggest a role of ion channels of the DEG/ENaC family for mechanosensation in different species and in baroreceptor reflex control in rats. We tested the hypothesis that ENaC within the cardiac sensory network are mandatory for mechanosensation. Experiments were performed in male Sprague-Dawley rats, isolated nodose ganglion cells with cardiac afferents and isolated vagus nerves. Epicardial delivery of the amiloride analogue benzamil intended to specifically inhibit ENaC presumably located on cardiac sensory afferents indeed blunted the mechanosensitive (i. e., sympathoinhibition by intravenous volume loading [-32% and -42% in treated groups vs. -67% in controls; n = 7 each; p < 0.05]) as well as-though to a lesser extent-the 5-HT(3)-mediated chemosensitive cardiorenal reflex in vivo in a dose-dependent manner. Using patch clamp technique, however, it turned out that neither amiloride nor benzamil influenced mechanically induced currents in ganglion nodosum cells in vitro, stimulated by hypoosmotic stress. The unspecific stretch activated ion channel blocker gadolinium completely abolished mechanically induced currents, indicating respective cells were mechanosensitive. In isolated vagus nerves benzamil impaired action potentials obtained by electrical stimulation (C-spike amplitude [-33%]; latency [+12%]; n = 8; p < 0.05). Our findings at least cast doubt on ENaC exclusively playing a specific role as mechanotransducers within the cardiac sensory network. Other ion channels might be involved. Furthermore the observed findings in vivo could also be due to unspecific disturbance of afferent signal conduction.

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

Brain sodium channels and central sodium-induced increases in brain ouabain-like compound and blood pressure.

OBJECTIVE: To assess the role of benzamil-sensitive sodium channels in the increases in brain ouabain-like compounds (OLC) and in blood pressure by cerebrospinal fluid (CSF) Na+. METHODS: Artificial CSF (aCSF) or Na+-rich (0.8 mol/l Na+) aCSF, either alone or combined with benzamil (at 1.2 and 4.0 microg/kg per h), were infused intracerebroventricularly (i.c.v.) at 5 microl/h to Wistar rats for 14 days and the effects on the brain and peripheral OLC and blood pressure were studied. OLC content was measured by enzyme-linked immunosorbent assay. RESULTS: In Wistar rats infused i.c.v. with aCSF, benzamil did not affect blood pressure or brain and peripheral OLC concentrations. I.c.v. infusion of Na+-rich aCSF increased systolic blood pressure (140 +/- 4 mmHg compared with 119 +/- 3 mmHg; P < 0.05). Benzamil fully blocked this increase. Na+-rich aCSF increased hypothalamic (23 +/- 3 ng/g tissue compared with 10 +/- 1 ng/g tissue; P < 0.05) and pituitary (233 +/- 35 ng/g tissue compared with 62 +/- 7 ng/g tissue; P < 0.05) contents of OLC. In contrast, Na+-rich aCSF decreased OLC in the adrenal gland (7 +/- 1 ng/g tissue compared with 21 +/- 3 ng/g tissue; P < 0.05) and plasma (0.5 +/- 0.04 ng/ml compared with 0.7 +/- 0.08 ng/ml; P < 0.05). Benzamil inhibited these responses of OLC to CSF sodium in a dose-related manner. CONCLUSIONS: These findings suggest that benzamil-sensitive brain sodium channels mediate the increase in brain OLC and the subsequent hypertension induced by increased CSF Na+.

[3H]-DA release evoked by low pH medium and internal H+ accumulation in rat hypothalamic synaptosomes: involvement of calcium ions.

The pH fluctuations have been often interpreted as an insufficient regulation or as a consequence of the onset of pathological events, such as ischemia, in which a significant decrease in pH levels occurs. Neurotransmitter release appears to be affected by pH drop significantly. In this study, we investigated the effect of an extracellular and an intracellular acidification on tritiated dopamine release ([3H]-DA release), from superfused rat hypothalamic synaptosomes. When compared to basal release, extracellular acidification, due to a reduction in the external pH of the nominally carbonic-free superfusion media, provoked a significant increase in [3H]-DA release that showed a sensitiveness to calcium omission. Intraterminal acidification, obtained blocking the Na(+)/H(+) exchanger by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and 5-(N,N-dimethyl)-amiloride (DMA), induced a significant increase in [3H]-DA outflow which occurred in a calcium-dependent manner (80% inhibition in absence of calcium from superfusion media). To further promote an intraterminal acidification through a H(+) inner accumulation, the proton ionophore nigericin was used. At every dose employed (10 microM), this compound induced a significant increase in [3H]-DA outflow, compared to basal release. Nigericin-evoked [3H]-DA release showed a 50% decrease when calcium was omitted from superfusion media. When BAPTA-AM, a chelator of intracellular calcium, was added, nigericin-evoked [3H]-DA was completely abolished. These data indicate that [3H]-DA release can be induced by extracellular acidification due to a lowering of external pH and by an intraterminal acidification due to an internal proton accumulation. The mechanism that can trigger this exocytotic process appears to depend on calcium presence, and in particular, on an increased intraterminal calcium availability.

Brain sodium channels mediate increases in brain "ouabain" and blood pressure in Dahl S rats.

Central infusions of benzamil prevent/reverse salt-induced hypertension in genetic models of salt-sensitive hypertension. Benzamil acts by blockade of ion--presumably sodium--channels. In the present study, we assessed in Dahl salt-sensitive (S) rats on high salt intake whether these channels mediate increases in brain "ouabain" and, thereby, hypertension. Intracerebroventricular (icv) infusions of a low (1.2 microg/kg per hour) or high (4.0 microg/kg per hour) dose of benzamil were given to Dahl S rats on high salt diet (1370 micromol Na+/g food) for 2 or 4 weeks. "Ouabain" content was measured using a specific enzyme-linked immunosorbent assay (ELISA). Systolic blood pressure (BP) in Dahl S rats on high salt for 4 weeks increased markedly (188+/-10 versus 128+/-4 mm Hg, n=8, P<0.05). Benzamil fully blocked this increase (131+/-7 mm Hg after the high dose of benzamil). Hypothalamic and pituitary "ouabain" increased significantly (22+/-7 versus 12+/-3 and 151+/-38 versus 69+/-6 ng/g tissue, respectively, P<0.05) in Dahl S rats on high salt versus regular salt diet for 2 weeks. Benzamil blocked these increases of brain "ouabain" to high salt intake. Similarly, high salt intake for 4 weeks increased hypothalamic (18+/-2 versus 13+/-1 ng/g tissue, P<0.05) and pituitary (183+/-30 versus 78+/-8 ng/g tissue, P<0.05) "ouabain." Benzamil also inhibited these increases of brain "ouabain." Both hypothalamic and pituitary "ouabain" showed significant positive correlations with BP. In contrast, high salt intake did not affect "ouabain" levels in the adrenal gland or plasma in Dahl S rats on high salt for either 2 or 4 weeks. These findings indicate that in Dahl S rats high salt intake only increases brain and not peripheral "ouabain" and that benzamil-blockable brain sodium channels mediate the increases in brain "ouabain" and the subsequent hypertension.

Role of intracellular calcium in the spermicidal action of 2',4'-dichlorobenzamil, a novel contact spermicide.

The Na+-Ca2+ exchanger and Ca2+-ATPase pumps reported to be present on the sperm membrane are responsible for maintaining the intracellular Ca2+ concentration that is involved in regulation of sperm function. We have investigated the role of intracellular Ca2+ in the presence of 2',4'-dichlorobenzamil hydrochloride (benzamil), a Na+-Ca2+ exchange inhibitor, on human sperm motility. The mechanism of the complementary spermicidal action produced by a combination of benzamil and propranolol on human spermatozoa has been investigated also. When administered alone benzamil and propranolol produced a dose- and time-dependent decrease in motility of sperm in ejaculated semen and spermatozoa separated from semen. A combination of benzamil and propranolol exhibited a complementary spermicidal action, thereby resulting in dose reduction of both drugs for obtaining total immotility within 1 min of administration. An increase in the intracellular Ca2+ level was found to contribute to the spermicidal activity. Inhibition of the Na+-Ca2+ exchange system on sperm membrane by benzamil and membrane stabilization by propranolol resulted in accumulation of Ca2+ inside the sperm cells. When the two drugs were used in combination the time required for the total loss of motility of spermatozoa was significantly reduced due to a similar mechanism of action of both drugs.

Amiloride derivatives are potent blockers of KATP channels.

In cardiomyocytes sarcolemmal KATP channels open massively when the cytosolic [ATP] drops into the range of tens of micromolar, as during acute ischemia. The diuretic drug amiloride and related derivatives are well established as drugs blocking the Na+/H+- and the Na+/Ca2+-exchange, protecting the ischemic heart. Herein, the blocking action of amiloride and its derivatives 2',4'-dichlorobenzamil (DCB) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) on KATP channels was tested. In inside-out patches of mouse cardiac myocytes, amiloride, DCB, and EIPA reversibly blocked the KATP channels with the IC50 values 102, 1.80, and 2.14 micromol/l (-80 mV), respectively. Similar IC50 values were obtained in recombinant channels when coexpressing the KIR6.2 subunit with one of the sulfonylurea receptors SUR1 and SUR2A. All three drugs also blocked currents generated by the C-terminus deletion mutant KIR6.2delta26 in the absence of SUR. Amiloride blocked outward currents more effectively than inward currents whereas the block by DCB and EIPA was voltage independent. In cardiomyocytes, also whole-cell IKATP was blocked by the three drugs. In conclusion, amiloride, EIPA, and DCB block the pore-forming KIR6.2 subunit of cardiac KATP channels with higher potency than the Na+/H+- and the Na+/Ca2+-exchange, precluding a specific block of the exchanges under ischemic conditions.

Na(+)-dependent pH regulation by the amitochondriate protozoan parasite Giardia intestinalis.

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.