Takeshi Katsuragi

Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle.

Excessive salt intake is a major risk factor for hypertension. Here we identify the role of Na+/Ca2+ exchanger type 1 (NCX1) in salt-sensitive hypertension using SEA0400, a specific inhibitor of Ca2+ entry through NCX1, and genetically engineered mice. SEA0400 lowers arterial blood pressure in salt-dependent hypertensive rat models, but not in other types of hypertensive rats or in normotensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increases arterial blood flow, indicating peripheral vasodilation. SEA0400 reverses ouabain-induced cytosolic Ca2+ elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice have low salt sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle are hypersensitive to salt. SEA0400 lowers the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca2+ entry through NCX1 in arterial smooth muscle and suggest that NCX1 inhibitors might be useful therapeutically.

Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400

SEA0400 is a potent and selective Na+/Ca2+ exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na+i-dependent 45Ca2+ uptake and whole-cell Na+/Ca2+ exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited 45Ca2+ uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the α-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an α-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na+-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na+-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.

3 possible coupling of postjunctional ATP release and transmitters' receptor stimulation in smooth muscles

The nature of ATP release from mainly smooth muscles of guinea-pig was evaluated with KCl and agonists for different kinds of receptors. In ileal longitudinal muscles, amounts of net ATP release by ACh and bethanechol (1-10 microM) were much larger (about 10 fold) than that by other drugs, e.g., histamine, 5-hydroxytryptamine, prostaglandin-F2 alpha, substance P and bradykinin, including KC1, although differences between contractions of the tissue evoked by test drugs were approximately 1.5 times at most. The ATP release, as well as the contraction, evoked by ACh or bethanechol was markedly reduced by atropine (0.3 microM), thus, indicating primarily postjunctional release of ATP. The remarkable ATP release from vas deferens by norepinephrine (NE), but not by substance P, was abolished almost completely by prazosin (0.3 microM). Increases in intracellular Ca2+ and subsequent contraction in the ileal tissue were produced by ATP and these responses were fully antagonized by nifedipine (0.1 microM). These findings provide evidence that the drugs-stimulated ATP release from smooth muscles does not result from contractility of muscles, but is substantially elicited only by stimulation of neurotransmitter (NE or ACh) receptors, suggesting the existence of the receptor-stimulus-postjunctional ATP release coupling. The released ATP may contribute, in part, to the muscle contractility via increase of Ca2(+)-influx, presumably, in a manner related to the voltage-gated Ca2(+)-channels.

Antagonism by nifedipine of contraction and Ca2+ ‐influx evoked by ATP in guinea‐pig urinary bladder

1 The effects of Ca2+‐antagonists, especially nifedipine, on contraction and increase of intracellular Ca2+ (Fura‐2/AM method) evoked by ATP were evaluated in a thin outer layer segment of guinea‐pig urinary bladder. 2 The ATP‐evoked contraction was markedly inhibited by dihydropyridine‐type Ca2+‐antagonists, such as nifedipine and nitrendipine, but not by D‐600, ω‐conotoxin and tetramethrin. 3 This antagonism by nifedipine of ATP‐evoked contractions was competitive from the Schild plot analysis, the pA2 value being 8.23. The reduction of ATP‐evoked contraction by nifedipine (0.1 μm) was fully reversed by administration of Bay K 8644 (0.1 μm). 4 ATP (100 μm) caused an increase of fluorescence brightness after loading Fura‐2/AM, which was coupled with a contraction of the bladder. Both the contraction and the elevation of intracellular Ca2+ evoked by the nucleotide were completely antagonized by nifedipine. 5 These results suggest that ATP may activate the dihydropyridine‐sensitive, voltage‐dependent Ca2+‐channels in a direct or indirect fashion and, thereby, elicit a contraction of the bladder.

Involvement of adenosine receptor activities in aggressive responses produced by clonidine in mice

A behavioral study was made of the mechanisms underlying the aggressive behavior induced by high doses of clonidine in mice. The frequency of clonidine-induced aggressive responses such as attacking and biting was increased dose-dependently from 10 to 50 mg/kg. Aggressive behavior induced by clonidine at doses of 10–30 mg/kg was potentiated under conditions of isolation and food deprivation for 24 h. Clonidine (30 mg/kg)-induced aggressive behavior was attenuated by adenosine (10 mg/kg IP) or dipyridamole (10 mg/kg IP), but markedly antagonized by combined pretreatment with both drugs. The behavior was strongly reduced by potent adenosine analogs, such as N6-cyclohexyl adenosine (CHA, 0.1 and 0.2 mg/kg IP) and N6-(l-phenyl isopropyl) adenosine (l-PIA, 0.2 mg/kg IP), but conversely was potentiated by phentolamine (10 mg/kg IP) or theophylline (10 mg/kg IP). Diazepam (2.5 mg/kg IP) and Ro 15-1788 (2.5 mg/kg IP), a benzodiazepine receptor antagonist, also blocked the aggressive behavior. The inhibition by CHA (0.2 mg/kg IP) or diazepam (2.5 mg/kg) of clonidine-induced aggression was not antagonized by additional pretreatment with bicuculline (2 mg/kg IP). The aggressive response to apomorphine (8 mg/kg IP) was not affected by those drugs which inhibited the response to clonidine. The results suggest that the aggressive behavior evoked by high doses of clonidine, but not that by apomorphine, involves a blockade of adenosine receptors.

Comparison of Effects of Nitric Oxide Synthase (NOS) Inhibitors on Plasma Nitrite/Nitrate Levels and Tissue NOS Activity in Septic Organs

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar‐King rats. The rats treated with the three iNOS inhibitors (N‐(3‐(aminomethyl)benzyl)acetamidine (1400W), (1S, 5S, 6R, 7R)‐2‐aza‐7‐chloro‐3‐imino‐5‐methylbicyclo [4.1.0] heptane hydrochloride (ONO‐1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose‐dependent decreases in plasma NOx levels and NOS activity in the lungs. The non‐selective NOS inhibitor (NG‐methyl‐L‐arginine (L‐NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.

The clonidine-induced self-injurious behavior of mice involves purinergic mechanisms

Mode of action of clonidine involved in self-injurious behavior was assessed in mice. Single injection of clonidine (50 mg/kg, IP) evoked a self-biting which occurred more frequently under the condition of isolation and food-deprivation for 24 hr. The clonidine-induced self-biting was not reduced, but rather potentiated by pretreatment with phentolamine (10 mg/kg). This behavior was enhanced by theophylline (20 mg/kg) but was inhibited to some extent by adenosine (10 mg/kg) or dipyridamole (10 mg/kg). In addition, the self-injurious behavior was completely antagonized by combined pretreatment with adenosine (10 mg/kg) and dipyridamole (10 mg/kg), and by potent adenosine agonists, such as N6-(L-phenylisopropyl) adenosine (0.2 mg/kg) and N6-cyclohexyladenosine (0.2 mg/kg). These results, therefore, suggest that the clonidine-induced self-biting could be substantially attributed to adenosine A1-receptor blockade as documented for pharmacological property of theophylline in the brain.

Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and α,β‐methylene ATP in guinea‐pig urinary bladder

1 The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea‐pig urinary bladder and compared with those evoked by ATP and αβ‐methylene ATP (a P2‐purinoceptor agonist). 2 AP4A, ATP and α,β‐methylene ATP produced concentration‐dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate‐6‐azophenyl‐ 2′‐4′‐disulphonic acid), 0.3–30 μm, a P2X‐purinoceptor antagonist, and suramin, 1–300 μm, a P2‐purinoceptor antagonist in a concentration‐dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and α,β‐methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3 AP4A (10 μm) and ATP (100 μm) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of α,β‐methylene ATP (1 μm). 4 In fluorescence experiments with fura‐2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L‐type Ca2+ channel blocker. 5 These findings suggest that P2X‐purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea‐pig urinary bladder, and that the AP4A‐evoked contraction results from Ca2+ influx.

Modulation of P2X receptors in dorsal root ganglion neurons of streptozotocin-induced diabetic neuropathy

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

Calcium antagonistic action involved in vasodilation by brovincamine.

Possible mode of vasodilative action of brovincamine was assessed in isolated cardiovascular preparations in comparison with verapamil and papaverine. Brovincamine (IC50: 1.2 x 10(-5) M), verapamil (IC50: 3.5 x 10(-7) M) or papaverine (IC50: 2.5 x 10(-5) M) caused a dose-dependent relaxation of potassium (30 mM)-contracture in the rabbit pulmonary arterial segment. This relaxation by verapamil or brovincamine, but not by papaverine, was antagonized by increasing external Ca2+ concentration to 12.4mM. Duration of slow action potentials of partially depolarized guinea-pig papillary muscle was reduced by 14 min exposure to brovincamine (5 x 10(-5) M) or verapamil (10(-5) M. These results suggest that brovincamine produces a vasodilation via a slow Ca2+-channel blockade.