The plasma renin test reveals the contribution of body sodium-volume content (V) and renin-angiotensin (R) vasoconstriction to long-term blood pressure.

Body sodium works together with the plasma renin-angiotensin system to ensure adequate blood flow to the tissues. Body sodium content determines the extracellular fluid (ECF) volume ensuring that, with each heart beat, a sufficient volume of fluid is delivered into the arterial space. At the same time the kidneys monitor ECF volume and blood pressure (BP), so that the juxtaglomerular cells can adjust their net secretion rate of renin to maintain an appropriate plasma renin activity (PRA) level. Plasma renin produces angiotensin II (Ang II) to constrict the arterioles and thereby ensure sufficient BP to deliver an appropriate rate of flow for cardiovascular homeostasis. The low renin, sodium-volume dependent (V) form of essential hypertension occurs whenever body sodium content increases beyond the point where plasma renin-angiotensin vasoconstrictor activity is turned off. In contrast, medium to high renin (R) hypertension occurs when too much renin is secreted relative to the body sodium content. Thus, BP = V × R. This volume-vasoconstriction dual support of long-term hypertension is validated by the fact that all effective long-term antihypertensive drug types are either (i) natriuretic to reduce body salt and volume content (anti-V), or (ii) antirenin to reduce or block the activity of the circulating renin-angiotensin system (anti-R). The PRA test defines the relative participation of the concurrent volume and vasoconstrictor factors. In the hypertensive patient PRA testing can guide initiation, addition or subtraction of anti-V or anti-R antihypertensive drug types to thereby improve BP control and prognosis while reducing drug type usage and cost.

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition.

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.

An ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein.

It is suggested that an ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein. We reported that glibenclamide, an ATP-sensitive potassium channel blocker, accelerated dose-dependent secretion of renal kallikrein in sliced kidney cortex and in vivo in rats. In vehicle-treated normal Brown- Norway-Kitasato (nBN-Ki) rats, the administration of glibenclamide increased urinary kallikrein secretion, but changed neither the systolic blood pressure nor the urinary sodium on low (0.3%) NaCl diets. Although on high (8%) NaCl diets, the systolic blood pressure of the nBN-Ki rats administrated glibenclamide was significantly lower (P<0.05). The urinary levels of kallikrein and sodium of the nBN-Ki rats administrated glibenclamide were significantly increased (P<0.05, glibenclamide vs. vehicle). A similar result was obtained with a kidney-selective ATP-sensitive potassium blocker, N,N'-dicyclohexyl-4-morpholinecarboxamidine (U18177), in SD rats. Mutant kininogen-deficient Brown-Norway Katholiek (muBN-Ka) rats fed high (8%) NaCl diets showed an increase in urinary kallikrein levels, but showed neither hypotensive nor natriuretic actions by glibenclamide. A bradykinin B(2) receptor antagonist, 8-[3-[N-(E)-3-(6-acetamidopyridin-3-yl) acryloylglyycyl]-N-methylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline (FR173657), which was administrated to SD rats, together with glibenclamide, abolished the hypotensive and natriuretic effects of glibenclamide in high-sodium (8%NaCl) hypertension, despite an accelerated secretion of urinary kallikrein. Therefore, these results indicate that glibenclamide, an ATP-sensitive potassium channel blocker suppressed sodium-induced hypertension through sodium excretion from the kidney resulting from accelerated secretion of urinary kallikrein.

Hyperforin--a key constituent of St. John's wort specifically activates TRPC6 channels.

Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, is the main active principle of St. John's wort extract responsible for its antidepressive profile. Hyperforin inhibits the neuronal serotonin and norepinephrine uptake comparable to synthetic antidepressants. In contrast to synthetic antidepressants directly blocking neuronal amine uptake, hyperforin increases synaptic serotonin and norepinephrine concentrations by an indirect and yet unknown mechanism. Our attempts to identify the molecular target of hyperforin resulted in the identification of TRPC6. Hyperforin induced sodium and calcium entry as well as currents in TRPC6-expressing cells. Sodium currents and the subsequent breakdown of the membrane sodium gradients may be the rationale for the inhibition of neuronal amine uptake. The hyperforin-induced cation entry was highly specific and related to TRPC6 and was suppressed in cells expressing a dominant negative mutant of TRPC6, whereas phylogenetically related channels, i.e., TRPC3 remained unaffected. Furthermore, hyperforin induces neuronal axonal sprouting like nerve growth factor in a TRPC6-dependent manner. These findings support the role of TRPC channels in neurite extension and identify hyperforin as the first selective pharmacological tool to study TRPC6 function. Hyperforin integrates inhibition of neurotransmitter uptake and neurotrophic property by specific activation of TRPC6 and represents an interesting lead-structure for a new class of antidepressants.

Lithium normalizes elevated intracellular sodium.

BACKGROUND: Both mania and bipolar depression are characterized by elevations of intracellular sodium concentrations. This observation has been purported to be central to the pathophysiology of abnormal moods in bipolar illness. Reduction of sodium influx is a proposed shared mechanism of action of effective mood stabilizers, but direct documentation of this effect for lithium has never been demonstrated. METHODS: Flame spectroscopic determinations of intracellular sodium concentration were performed in the human glioma cell line, LN292, after treatment with the sodium pump inhibitor, ouabain, and co-treatment with ouabain and lithium. RESULTS: Ouabain 0.1 microM doubles the intracellular sodium concentration after 3 days. Pretreatment with lithium 1 mM for 1 week normalizes intracellular sodium. CONCLUSION: This is the first demonstration that lithium can normalize abnormally elevated intracellular sodium levels. This may be an important mechanism of lithium action.

Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells.

BACKGROUND: GLP-1 is secreted into the circulation after food intake. The main biological effects of GLP-1 include stimulation of glucose dependent insulin secretion and induction of satiety feelings. Recently, it was demonstrated in rats and humans that GLP-1 can stimulate renal excretion of sodium. Based on these data, the existence of a renal GLP-1 receptor (GLP-1R) was postulated. However, the exact localization of the GLP-1R and the mechanism of this GLP-1 action have not yet been investigated. METHODS: Primary porcine proximal tubular cells were isolated from porcine kidneys. Expression of GLP-1R was measured at the mRNA level by quantitative RT-PCR. Protein expression of GLP-1R was verified with immunocytochemistry, immunohistochemistry and Western blot analysis. Functional studies included transport assessments of sodium and glucose using three different GLP-1 concentrations (200 pM, 2 nM and 20 nM), 200 pM exendin-4 (GLP-1 analogue) and an inhibitor of the dipeptidylpeptidase IV (DPPIV) enzyme (P32/98 at 10 microM). Finally, the expression of NHE3, the predominant Na(+)/H(+) exchanger in proximal tubular cells, was also investigated. RESULTS: GLP-1R, NHE3 and DPPIV were expressed at the mRNA level in porcine proximal tubular kidney cells. GLP-1R expression was confirmed at the protein level. Staining of human and pig kidney cortex revealed that GLP-1R was predominantly expressed in proximal tubular cells. Functional assays demonstrated an inhibition of sodium re-absorption with GLP-1 after 3 h of incubation. Exendin-4 and GLP-1 in combination with P32/98 co-administration had no clear influence on glucose and sodium uptake and transport. CONCLUSION: GLP-1R is functionally expressed in porcine proximal tubular kidney cells. Addition of GLP-1 to these cells resulted in a reduced sodium re-absorption. GLP-1 had no effect on glucose re-absorption. We conclude that GLP-1 modulates sodium homeostasis in the kidney most likely through a direct action via its GLP-1R in proximal tubular cells.

Involvement of oxidative stress induction in Na+ toxicity and its relation to the inhibition of a Ca2+ -dependent but calcineurin-independent mechanism in Saccharomyces cerevisiae.

Uridine 5'-hexadecylphosphate (UMPC16) inhibited the growth of Saccharomyces cerevisiae under a hypersaline stress condition with Na+ more strongly than the calcineurin inhibitor cyclosporine A (CsA). Additional Ca2+ supplementation similarly suppressed the inhibitory activities of UMPC16 and CsA on yeast cell growth in a medium with Na+. UMPC16, but not CsA, accelerated mitochondrial reactive oxygen species (ROS) generation in combination with Na+, suggesting its inhibition of a Ca2+ -dependent but calcineurin-independent mechanism for protection against Na+ toxicity.

A dynamic model of excitation-contraction coupling during acidosis in cardiac ventricular myocytes.

Acidosis in cardiac myocytes is a major factor in the reduced inotropy that occurs in the ischemic heart. During acidosis, diastolic calcium concentration and the amplitude of the calcium transient increase, while the strength of contraction decreases. This has been attributed to the inhibition by protons of calcium uptake and release by the sarcoplasmic reticulum, to a rise of intracellular sodium caused by activation of sodium-hydrogen exchange, decreased calcium binding affinity to Troponin-C, and direct effects on the contractile machinery. The relative contributions and concerted action of these effects are, however, difficult to establish experimentally. We have developed a mathematical model to examine altered calcium-handling mechanisms during acidosis. Each of the alterations was incorporated into a dynamical model of pH regulation and excitation-contraction coupling to predict the time courses of key ionic species during acidosis, in particular intracellular pH, sodium and the calcium transient, and contraction. This modeling study suggests that the most significant effects are elevated sodium, inhibition of sodium-calcium exchange, and the direct interaction of protons with the contractile machinery; and shows how the experimental data on these contributions can be reconciled to understand the overall effects of acidosis in the beating heart.

Protective effect of endogenous beta-adrenergic tone on lung fluid balance in acute bacterial pneumonia in mice.

Some investigators have reported that endogenous beta-adrenoceptor tone can provide protection against acute lung injury. Therefore, we tested the effects of beta-adrenoceptor inhibition in mice with acute Escherichia coli pneumonia. Mice were pretreated with propranolol or saline and then intratracheally instilled with live E. coli (10(7) colony-forming units). Hemodynamics, arterial blood gases, plasma catecholamines, extravascular lung water, lung permeability to protein, bacterial counts, and alveolar fluid clearance were measured. Acute E. coli pneumonia was established after 4 h with histological evidence of acute pulmonary inflammation, arterial hypoxemia, a threefold increase in lung vascular permeability, and a 30% increase in extravascular lung water as an increase in plasma catecholamine levels. beta-Adrenoceptor inhibition resulted in a marked increase in extravascular lung water that was explained by both an increase in lung vascular permeability and a reduction in net alveolar fluid clearance. The increase in extravascular lung water with propranolol pretreatment was not explained by an increase in systemic or vascular pressures. The increase in lung vascular permeability was explained in part by anti-inflammatory effects of beta-adrenoceptor stimulation because plasma macrophage inflammatory protein-2 levels were higher in the propranolol pretreatment group compared with controls. The decrease in alveolar fluid clearance with propranolol was explained by a decrease in catecholamine-stimulated fluid clearance. Together, these results indicate that endogenous beta-adrenoceptor tone has a protective effect in limiting accumulation of extravascular lung water in acute severe E. coli pneumonia in mice by two mechanisms: 1) reducing lung vascular injury and 2) upregulating the resolution of alveolar edema.

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss).

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.

Effectiveness of levobetaxolol and timolol at blunting retinal ischaemia is related to their calcium and sodium blocking activities: relevance to glaucoma.

Glaucoma is a chronic optic neuropathy in which retinal ganglion cells die over a number of years. The initiation of the disease and its progression may involve an ischaemic-like insult to the ganglion cell axons caused by an alteration in the quality of blood flow. Thus, to effectively treat glaucoma it may be necessary to counteract the ischaemic-like insult to the region of the optic nerve head. Studies on the isolated optic nerve suggest that substances that reduce the influx of sodium would be particularly effective neuroprotectants. Significantly, of the presently used antiglaucoma substances, only beta-blockers can reduce sodium influx into cells. Moreover, they also reduce the influx of calcium and this would be expected to benefit the survival of insulted neurones. Betaxolol is the most effective antiglaucoma drug at reducing sodium/calcium influx. Our electroretinographic data indicated that topical application of levobetaxolol to rats attenuated the effects of ischaemia/reperfusion injury. Timolol was also effective but to a lesser extent. Based on these data we conclude that beta-blockers may be able to blunt ganglion cell death in glaucoma, and that levobetaxolol may be a more effective neuroprotectant than timolol because of its greater capacity to block sodium and calcium influx.

Momordica charantia extracts inhibit uptake of monosaccharide and amino acid across rat everted gut sacs in-vitro.

The inhibitory effects of Momordica charantia extracts were studied on the uptake of glucose and tyrosine across rat everted gut sacs in vitro. The aqueous extract of the plant was found to inhibit primarily the uptake of glucose in a dose-dependent manner. Uptake of tyrosine was affected at high substrate concentrations only. The extract was also found to decrease the absorptive capacity of fluid across the small intestine and sodium ions. It is hypothesized that the effects of Momordica could involve a washout of glucose from the blood stream.

Role of basolateral membrane conductance in the regulation of transepithelial sodium transport across frog skin.

Circuit analyses of the principal cell compartment of frog skin ( Rana temporaria and R. esculenta) were made using microelectrode measurements under short-circuit conditions and with the aid of the Na(+) channel blocker amiloride. Under control conditions, intracellular potential ranged between -65 and -5 mV, and the conductances of the apical and basolateral membranes were related directly to the short-circuit current and inversely to the cellular potential. Blockade of apical Na(+) uptake by amiloride hyperpolarized the cells to nearly the same value, irrespective of the potential under transporting conditions. Under these conditions, basolateral membrane conductance increased greatly, which led to paradoxical reactions of the transepithelial Na(+) transport at lower concentrations of amiloride. The half-maximal inhibitory concentration of amiloride estimated from the response of the apical membrane conductance (99+/-10 nM) was about 5 times lower than the value derived from transepithelial current or conductance in the same tissues. The results are discussed in the context of the importance of the membrane potential for acute control of membrane conductance and transepithelial transport.

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification.

In this study, we test the hypothesis that in newborn hearts (as in adults) hypoxia and acidification stimulate increased Na(+) uptake, in part via pH-regulatory Na(+)/H(+) exchange. Resulting increases in intracellular Na(+) (Na(i)) alter the force driving the Na(+)/Ca(2+) exchanger and lead to increased intracellular Ca(2+). NMR spectroscopy measured Na(i) and cytosolic Ca(2+) concentration ([Ca(2+)](i)) and pH (pH(i)) in isolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. After Na(+)/K(+) ATPase inhibition, hypoxic hearts gained Na(+), whereas normoxic controls did not [19 +/- 3.4 to 139 +/- 14.6 vs. 22 +/- 1.9 to 22 +/- 2.5 (SE) meq/kg dry wt, respectively]. In normoxic hearts acidified using the NH(4)Cl prepulse, pH(i) fell rapidly and recovered, whereas Na(i) rose from 31 +/- 18.2 to 117.7 +/- 20.5 meq/kg dry wt. Both protocols caused increases in [Ca](i); however, [Ca](i) increased less in newborn hearts than in adults (P < 0.05). Increases in Na(i) and [Ca](i) were inhibited by the Na(+)/H(+) exchange inhibitor methylisobutylamiloride (MIA, 40 microM; P < 0.05), as well as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data support the hypothesis that in newborn hearts, like adults, increases in Na(i) and [Ca](i) during hypoxia and after normoxic acidification are in large part the result of increased uptake via Na(+)/H(+) and Na(+)/Ca(2+) exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca](i) is less in newborn than adult hearts.

Localization and characterization of phenamil-sensitive Na+ influx in isolated rainbow trout gill epithelial cells.

Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na(+) uptake. Analysis of the mechanism(s) of (22)Na(+) influx was performed in control and acid-activated (addition of 10 mmoll(-1) proprionic acid) cells using a variety of Na(+) transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml(-1) Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na(+) uptake and that influx was unchanged during either bafilomycin (10 nmoll(-1)) treatment or internal acidification with addition of proprionic acid (10 mmoll(-1)). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml(-1) interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na(+) influx. Further separation of the MR fraction into PNA(+) and PNA(-) fractions using magnetic separation demonstrated that only the PNA(-) cells (alpha-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated (22)Na(+) uptake. We confirm the coupling of a V-type H(+)-ATPase with phenamil-sensitive Na(+) uptake activity and conclude that high-density alpha-MR cells function in branchial Na(+) uptake in freshwater fish.

Angiotensin receptors contribute to blood pressure homeostasis in salt-depleted SHR.

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 micromol/kg), PD-123319 (0.12 micromol. kg(-1). min(-1)), [d-Ala(7)]ANG-(1-7) (10 and 100 pmol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of AT(2) and AT((1-7)) receptors significantly increased the blood pressure of losartan-treated SHR (+15 +/- 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of AT((1-7)) receptors (15 +/- 4 mmHg). On the other hand, sole blockade of AT(2) receptors in losartan-treated SHR increased mean arterial pressure by 8 +/- 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of AT((1-7)) receptors alone or combined blockade of AT((1-7)) and AT(2) receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+11 +/- 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [d-Ala(7)]ANG-(1-7) (+7 +/- 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-AT(1) receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of AT(2) and AT((1-7)) receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan.

Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

Effects of butyrate on active sodium and chloride transport in rat and rabbit distal colon.

Short chain fatty acids, particularly butyrate, stimulate electroneutral NaCl absorption from the colon. Their effect in colonic epithelia lacking basal electroneutral NaCl absorption is unknown. Butyrate is also reported to inhibit active Cl- secretion in the colon. The present studies were undertaken to investigate the inter-relationships between the effects of butyrate on active Na+ and Cl- transport in the colon. Studies were carried out in rabbit distal colon (known to have predominant electrogenic Na+ absorption), rat distal colon (characterised by electroneutral Na+ absorption), and hyperaldosteronaemic rat distal colon (characterised by electrogenic Na+ absorption). The effect of cholera toxin (CT) was also noted. Potential difference, short-circuit current (I(SC)) and fluxes of Na+ and Cl- were measured in stripped mucosa under voltage-clamp conditions. Butyrate stimulated electroneutral Na+ and Cl- absorption in distal colon of normal and salt-depleted rats, and stimulated Na+ absorption in rabbit distal colon. Amiloride (10(-4) M) or CT did not inhibit this process. In rabbit distal colon, stimulation of Na+ absorption by butyrate was not dependent on the presence of Cl- in the medium. Butyrate significantly decreased conductance, decreased flux of sodium from serosa to mucosa (particularly in rabbit distal colon), and decreased I(SC). Net Cl- secretion, induced by CT, was completely inhibited by butyrate. Stimulation of Na+ absorption was independent of exposure to CT. Bumetanide reversed net Cl- secretion to net absorption, but did not alter Na+ or Cl- fluxes in tissues exposed to butyrate. Thus butyrate stimulates active Na+ absorption in colonic epithelia, with or without expression of basal Na+-H+ exchange. Independently, butyrate inhibits active Cl- secretion induced by cAMP in these epithelia.

Basolateral PAR-2 receptors mediate KCl secretion and inhibition of Na+ absorption in the mouse distal colon.

Proteinase-activated receptor-2 (PAR-2) may participate in epithelial ion transport regulation. Here we examined the effect of mouse activating peptide (mAP), a specific activator of PAR-2, on electrogenic transport of mouse distal colon using short-circuit current (I(SC)) measurements. Under steady-state conditions, apical application of amiloride (100 microM) revealed a positive I(SC) component of 74.3 +/- 6.8 microA x cm(-2) indicating the presence of Na+ absorption, while apical Ba2+ (10 mM) identified a negative I(SC) component of 26.2 +/- 1.8 microA x cm(-2) consistent with K+ secretion. Baseline Cl- secretion was minimal. Basolateral addition of 20 microM mAP produced a biphasic I(SC) response with an initial transient peak increase of 11.2 +/- 0.9 microA x cm(-2), followed by a sustained fall to a level 31.2 +/- 2.6 microA x cm(-2) (n = 43) below resting I(SC). The peak response was due to Cl- secretion as it was preserved in the presence of amiloride but was largely reduced in the presence of basolateral bumetanide (20 microM) or in the absence of extracellular Cl-. The secondary decline of I(SC) was also attenuated by bumetanide and by Ba2+, indicating that it is partly due to a stimulation of K+ secretion. In addition, the amiloride-sensitive I(SC) was slightly reduced by mAP, suggesting that inhibition of Na+ absorption also contributes to the I(SC) decline. Expression of PAR-2 in mouse distal colon was confirmed using RT-PCR and immunocytochemistry. We conclude that functional basolateral PAR-2 is present in mouse distal colon and that its activation stimulates Cl- and K+ secretion while inhibiting baseline Na+ absorption.

Dehydroepiandrosterone sulfate (DHEAS) suppresses P2X purinoceptor-coupled responses in PC12 cells.

Some steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels in addition to their well-known genomic effects via intracellular steroid receptors. Such effects were found in GABA receptor, nicotinic receptors, yet not investigated in P2X purinoceptors. In this study, the effects of dehydroepiandrosterone sulfate on the P2 purinoceptor was investigated. Results show that dehydroepiandrosterone sulfate acutely inhibits P2X purinoceptor functions in PC12 cells. Dehydroepiandrosterone sulfate suppressed ATP-induced cytosolic free calcium concentration ([Ca(2+)](i)) rise, cytosolic free sodium concentration ([Na(+)](i)) rise, and dopamine secretion in the presence of external calcium, but had no effect on ATP-induced [Ca(2+)](i) rise in the absence of external calcium or on UTP-induced [Ca(2+)](i) rise in the absence or presence of external calcium. Our data show that dehydroepiandrosterone sulfate exerted its effect on P2X, but not on the P2Y purinoceptors found in PC12 cells. Estradiol and estrone have similar effects on P2X purinoceptor, but dehydroepiandrosterone and progesterone do not.