Functional role of Cl- channels in acidic pH-induced contraction of the aorta of spontaneously hypertensive and Wistar Kyoto rats

pH regulates various cellular functions. Previously, we have described that acidic pH produces depolarization and contraction in isolated aorta from spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats [Br. J. Pharmacol. 118 (1996) 485]. The aim of the present study was to investigate the involvement of Cl- channels in acidic pH-induced contraction. Changing the pH of the bathing solution from 7.4 to 6.5 induced a contraction in both SHR and WKY aorta, which was 127.50+/-13.32% and 79.27+/-0.94% of the 64.8 mM KCl-induced contraction, respectively. The acidic pH-induced contraction was partially inhibited by the voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (1 microM) and nifedipine (0.1 microM). The Cl- channel inhibitors, diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (0.5 mM), 9-anthracene chloride (0.5 mM), indanyloxyacetic acid (30 microM) and niflumic acid (3 microM) also inhibited the acidic pH-induced contraction and the degree of attenuation was comparable to that of VDCC blockers. DIDS, 9-anthracene chloride and niflumic acid at concentrations used to inhibit the acidic pH-induced contraction also inhibited the 10 microM phenylephrine-induced contraction partially, without affecting the 64.8 mM KCl-induced contraction, whereas both the contractions were inhibited by indanyloxyacetic acid with equal efficacy. Indanyloxyacetic acid but not DIDS, 9-anthracene chloride or niflumic acid inhibited the 24.8 mM KCl-induced contraction. Simultaneous measurement of cytosolic Ca2+ and tension showed that niflumic acid reversed the increase in intracellular Ca2+ level and inhibited the contraction caused by acidic pH. Similarly, acidic pH depolarized the cultured vascular smooth muscle cells from SHR and the depolarization was completely reversible after the administration of niflumic acid. All these results suggest that the activation of Cl- channels is an important mechanism underlying the depolarization and contraction induced by acidic pH in SHR and WKY aortas.     

Enhancement of intracellular Cl- concentrations induced by extracellular ATP in guinea pig ventricular muscle

We investigated effects of extracellular ATP on intracellular chloride activities ([Cl-]i) and possible contribution of the Cl--HCO3- exchange to this increase in [Cl-]i in isolated guinea pig ventricular muscles. The [Cl-]i and intracellular pH (pHi) were recorded in quiescent ventricular muscles using double-barreled ion-selective microelectrode techniques. MgATP at a concentration higher than 0.1 mM, induced an increase in [Cl-]i, and this increase in [Cl-]i was dependent on the concentration of ATP but not on the concentration of magnesium ions present in the perfusion solution. NaADP, but not NaAMP, at a concentration of 0.5 mM induced a similar increase in [Cl-]i as that induced by MgATP. However, the NaADP-induced increase in [Cl-]i was transient and gradually returned to the control level even though NaADP was continuously present. Furthermore, ATP also triggered a transient acidification of pHi, and both increases in [Cl-]i and intracellular H+ induced by ATP were prevented when preparations were pretreated with stilbene derivatives, SITS and DIDS, or perfused with a Cl--free solution. Our findings showed that the increased extracellular ATP concentrations might trigger an increase in [Cl-]i in ventricular muscles. In light of previous studies showing that cardiac ischemia induced increases in extracellular nucleotide concentrations and [Cl-]i in ventricular muscles, we propose that ischemia-induced accumulation of ATP concentration in the extracellular space may be an important factor to trigger increment of [Cl-]i during ischemic conditions.     

Strain-specific effects of acidic pH on contractile state of aortas from Wistar and Wistar Kyoto rats

The effects of acidosis were investigated on the resting and precontracted aortas from Wistar and Wistar Kyoto (WKY) rats. Decrease in pH from 7.4 to 6.5, having no effect on the resting tension of Wistar aorta, induced a marked contraction of WKY aorta. Acidic pH markedly relaxed the contraction to 300 nM phenylephrine in Wistar aorta, whereas in WKY aorta, it produced a biphasic response, an initial relaxation followed by potentiation of the contraction. In aortas loaded with fura 2-AM, phenylephrine caused an increase in intracellular Ca2+ ([Ca2+]i) and a contraction in both Wistar and WKY rats. pH 6.5 produced a decrease in [Ca2+]i to a near-basal level and almost abolished the phenylephrine-induced contraction in Wistar rat aorta. However, in WKY aorta, a biphasic response, an initial decline and later a recovery of [Ca2+]i level, was observed. Interestingly, at similar sustained [Ca2+]i, the contractile response to phenylephrine in WKY aorta was potentiated under acidic pH conditions. Acidic pH-induced inhibition of the contraction to phenylephrine was unaffected by iberiotoxin, 4-aminopyridine, and glibenclamide (Ca2+-activated, delayed rectifier and ATP-sensitive K+ channel inhibitors, respectively), in aortas from both Wistar and WKY. Decrease in extracellular pH was associated with a rapid fall in intracellular pH (pHi) and the intracellular acidification profile was not different in both strains. All these results show that acidic pH induces strain-specific inhibitory and excitatory effects on the contractile state of aortas from Wistar and WKY rats, respectively. The sustained and transient relaxant responses to acidic pH in Wistar and WKY aortas, respectively, are due to decrease in [Ca2+]i levels, but this decrease in [Ca2+]i is independent of the activation of K+ channels.     

Levetiracetam inhibits Na+-dependent Cl-/HCO3- exchange of adult hippocampal CA3 neurons from guinea-pigs

The novel anticonvulsant levetiracetam (LEV) was tested for effects on bioelectric activity and intracellular pH (pHi) regulation of hippocampal CA3 neurons from adult guinea-pigs. In 4-aminopyridine-treated slices, LEV (10-100 microm) reduced the frequency of action potentials and epileptiform bursts of CA3 neurons by 30-55%, while the shape of these potentials remained largely unchanged. Suppressive effects were reversed by an increase of pHi with trimethylamine (TMA). Using BCECF-AM-loaded slices, we found that LEV (10-50 microm) reversibly lowered neuronal steady-state pHi by 0.19 +/- 0.07 pH units in the presence of extracellular CO2/HCO3- buffer. In the nominal absence of extracellular CO2/HCO3- or in Na+-free CO2/HCO3(-)-buffered solution, LEV had no effect on steady-state pHi. Recovery of pHi subsequent to ammonium prepulses remained unchanged in the absence of CO2/HCO3- buffer, but was significantly reduced by LEV in the presence of CO2/HCO3- buffer. These findings show that LEV inhibits HCO3(-)-dependent acid extrusion, but has no effect on Na+/H+ exchange. LEV did not affect Na+-independent Cl-/HCO3- exchange because intracellular alkalosis upon withdrawal of extracellular Cl- remained unchanged. These data show that LEV at clinically relevant concentrations inhibits Na+-dependent Cl-/HCO3- exchange, lowers neuronal pHi, and thereby may contribute to its anticonvulsive activity.     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH

BACKGROUND AND PURPOSE

Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown.

EXPERIMENTAL APPROACH

Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown.

KEY RESULTS

ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar ‘dose’-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1.

CONCLUSIONS AND IMPLICATIONS

These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.     

Investigations of the dual contractile/relaxant properties showed by antioquine in rat aorta.

1. In the present study we assessed the activity of antioquine, a bisbenzyltetrahydroisoquinoline alkaloid isolated from Pseudoxandra sclerocarpa, by examining its effects on the contractile activity of rat isolated aorta, specific binding of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin to cerebral cortical membranes and the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta. 2. Contractions in rat aorta induced by high concentrations of KCl (80 mM) and noradrenaline (1 microM) were inhibited by antioquine in a concentration-dependent manner (0.1 microM- 300 microM). The alkaloid appeared more potent against KCl-induced contractions. This inhibitory effect was observed at both 37 degrees C and 25 degrees C. 3. Paradoxically, at the highest concentration tested (300 microM) antioquine induced a contractile response of similar magnitude in the presence and absence of extracellular calcium, at 37 degrees C. This activity was greatly attenuated at 25 degrees C. Antioquine-induced contractions were not inhibited by prazosin (0.1 microM), nifedipine (1 microM) or diltiazem (100 microM). On the contrary, prazosin and nifedipine slightly increased the contractions in the presence of extracellular calcium. Papaverine (100 microM) partially inhibited the contractile response to antioquine both in the presence and absence of extracellular calcium. 4. At 25 degrees C, in Ca(2+)-free solution, antioquine (300 microM) did not modify the contractile response (phasic and tonic) evoked by noradrenaline, but increased the phasic contraction induced by caffeine. At 37 degrees C, the contraction elicited by antioquine made it impossible to observe the noradrenaline-induced one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Okadaic acid, a protein phosphatase inhibitor, stimulates the activity of Na+/H+ and Na+-independent Cl–/HCO3 – exchangers in human lymphocytes

The cytosolic pH (pHi) regulatory mechanisms of peripheral blood human lymphocytes and the effect of okadaic acid on the activity of these mechanisms were studied by means of fluorescence imaging microscopy of 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF)-loaded individual cells. Lymphocytes recover from a CO(2)-induced acid load in an extracellular Na+-dependent, intracellular Cl- -independent fashion. This pHi recovery is highly sensitive to the anion exchange inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and minimally sensitive to the Na+/H+ exchange inhibitor amiloride, suggesting that it is mostly due to the action of a Na+-dependent HCO3- transporter. Extracellular Cl- and Na+ removal experiments point to the existence of a DIDS-sensitive Na+-independent Cl-/HCO3- exchanger. Preincubation with okadaic acid stimulates the pHi recovery rate from a CO2-induced acid load in the presence of DIDS (0.002 pHu/min vs. 0.065 pHu/min), but not in the presence of amiloride. Okadaic acid also accelerates the pHi elevation induced by Cl- removal (0.039 pHu/min vs. 0.067 pHu/min). In summary, these results indicate that okadaic acid stimulates the activity of Na+/H+ and Na+-independent Cl-/HCO3- exchangers, but has no effect on the activity of the Na+-dependent HCO3- transporter of human lymphocytes.     

Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats

Compared to sympathetic nervous system, the role of parasympathetic innervation on tone development, especially under diseased conditions, of the pulmonary artery is relatively unknown. In this study, the contractile effect of acetylcholine and the type(s) of muscarinic (M) receptor involved in the pulmonary artery (1st intralobar branch; endothelium-denuded, under resting tension) of the normotensive Wistar-Kyoto (WKY) and age-matched (male, 22-26 weeks old) Spontaneously hypertensive rats (SHR) were investigated. Cumulative administration of acetylcholine (> or =0.1 microM) caused a concentration-dependent increase in tension (antagonised by p-fluoro-hexahydro-sila-difenidol and 4-diphenylacetoxy-N-methylpiperidine, both are selective muscarinic M(3) receptor antagonists) and the magnitude of maximum contraction (expressed as % of 50 mM [K(+)](o)-induced contraction) was markedly enhanced in the presence of neostigmine (10 microM, an anti-cholinesterase) (acetylcholine 30 microM, SHR: 72% vs. 35%; WKY: 32% vs. 20%). In SHR only, acetylcholine-elicited contraction was suppressed by 1-[beta-[3-(4-Methoxyphenyl)-propoxyl]-4-methoxyphenethyl]-1H-imidazole (SK&F 96365, 1 microM), amiloride (500 microM), ethyl-isopropyl-amiloride (EIPA, 10 microM), 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R 7943, 5 microM), 2,4-dichlorobenzamil (10 microM), and an equal molar substitution of [Na(+)](o) (< or =30 mM) with choline or N-methyl-D-glucamine. In nominally [Ca(2+)](o)-free, EGTA (0.5 mM)-containing Krebs' solution, acetylcholine (> or =3 microM) only elicited a small contraction. In conclusion, muscarinic M(3) receptor activation is responsible for the pulmonary artery contraction induced by acetylcholine, with a greater magnitude observed in SHR. The exaggerated contraction in SHR is probably due to an influx of [Na(+)](o) through the Na(+)/H(+) exchanger and the store-operated channels (SOC) into smooth muscle cells. Elevation of cytosolic [Na(+)](i) subsequently leads to an influx of [Ca(2+)](o) through the reverse mode of the Na(+)/Ca(2+) exchanger seems to play a permissive role in mediating the exaggerated contractile response of acetylcholine recorded in the SHR.     

Evidence for the involvement of protein kinase C in acidic pH-induced contraction in spontaneously hypertensive rat aorta

This study was performed to test the hypothesis that activation of protein kinase C (PKC) is a mechanism underlying the acidic pH-induced contraction (APIC) in spontaneously hypertensive rat (SHR) aorta. Changing pH of the bathing solution from 7.4 to 6.5 induced a marked contraction of SHR aorta. PKC inhibitors, GF109203X and calphostin C markedly inhibited the APIC selectively, without having a marked effect on the KCl-induced contraction. Inhibitors of mitogen-activated protein kinase kinase, U0126 and PD98059 mildly but significantly attenuated the APIC. However, at the similar concentrations both U0126 and PD98059 inhibited the KCl-induced contraction in a manner similar to that observed in APIC. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC) markedly inhibited the APIC and the extent of inhibition by this compound was similar to that shown by PKC inhibitors. Whereas, U-73122 and propranolol, inhibitors of phosphatidylinositol-specific PLC and phosphatidate phosphohydrolase, respectively, had no affect on the APIC. A tyrosine kinase inhibitor, tyrphostin 23 and GF109203X inhibited the APIC in an additive manner, and together they abolished the contractile response. From all these results, it is suggested that a significant component of the contraction observed in response to acidosis in SHR aorta is dependent upon the activation of PKC that seems to be the downstream event of the activation of PC-PLC. Furthermore, PKC- and tyrosine kinase-dependent pathways underlying the APIC are independent of each other.     

Chloride efflux is involved in ET-1 and 5-HT-induced contraction in rabbit basilar artery

Chloride (Cl-) efflux induces depolarization and contributes to contraction of cerebral arteries. We tested the effect of endothelin-1 and 5-hydroxytryptamine on isometric tension in rabbit basilar artery by inhibition of Na+-K+-2Cl- co-transporter and Cl-/HCO3- exchanger to decrease Cl-, by decreasing extracellular Cl- concentration, and by blocking Cl- channels using Cl- channel inhibitors. We have made the following observations: (i)endothelin-1 and 5-hydroxytryptamine produced contraction in the normal Cl- Krebs-Henseleit bicarbonate solution (123 mM Cl-); (ii)inhibition of Na+-K+-2Cl- co-transporter with bumetanide abolished the contractions; (iii) bicarbonate-free solution with HEPES reduced contractions to 5-hydroxytryptamine and endothelin-1; (iv) substitution of extracellular Cl- with methanesulfonate acid (MS- 113 mM, Cl- 10 mM) enhanced peak contraction to 5-hydroxytryptamine and endothelin-1 and decreased plateau contraction to 5-hydroxytryptamine, but did not affect the plateau contraction to endothelin-1 and KCl; and (v) blockade of Ca2+-dependent Cl- channel with niflumic acid and non-selective Cl- channel with 5-nitro-2-(3-phenylpropylamino) benzoic acid and indanyloxyacetic acid-94, R- (+)-methylindazone (R- (+)-IAA-94)decreased contractions to endothelin-1 and 5-hydroxytryptamine.However, removal of endothelium attenuated the effect of Cl-channel inhibitors. In conclusion, Cl- channels and Cl- flux are involved in endothelin-1-induced and 5-hydroxytryptamine-induced contraction in rabbit basilar artery. Cl- channel blockers might exert additional effects by releasing vasodilatation agents from endothelial cells.     

Effect of high extracellular Ca(2+) levels in spontaneously hypertensive rat aorta.

The release of endothelial relaxing factors has been suggested to be important in modulating the inhibition of the contractile activity caused by the increase in extracellular Ca(2+) concentration in arterial tissue. Since the hypertensive process in spontaneously hypertensive rats (SHR) could be associated with the release of endothelial vasoconstrictor factors (mainly cyclooxygenase-dependent endoperoxides and endothelin-1), we studied the contractile responses to KCl, methoxamine and phenylephrine in different aorta ring preparations (intact, de-endothelized, 10(-5) M indomethacin-treated, 10(-6) M CGS-27830 [meso-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridine carboxylic acid anhydride]-treated, and treated simultaneously with 10(-5) M indomethacin and 10(-6) M CGS-27830) from SHR and normotensive Wistar Kyoto rats (WKY), at various Ca(2+) concentrations (1.25, 2.5, 5 and 10 mM) in the organ bath. In endothelium-intact preparations from WKY rats we observed a decrease in KCl, methoxamine and phenylephrine contractions with high Ca(2+) concentrations (5 and 10 mM), but in the endothelium-intact preparations from SHR, the increase in extracellular Ca(2+) concentration potentiated methoxamine contractions and caused no change in KCl and phenylephrine contractions. When the endothelium was disrupted in preparations from both WKY rats and SHR, we observed a decrease in KCl and methoxamine contractions with high Ca(2+) concentrations. The decrease in phenylephrine contractions caused by high Ca(2+) concentrations was clear in de-endothelized preparations from WKY rats but slight in de-endothelized preparations from SHR. In all indomethacin- and CGS-27830-treated preparations, and also in the preparations from WKY rats and SHR treated with both drugs, we observed a decrease in all the contractile responses with increased Ca(2+) concentration. Besides, there was a clear reduction in the responses of the alpha(1)-adrenoceptor agonists in the WKY and SHR preparations treated with both drugs. The results indicate that, in the hypertensive arteries, endothelium-derived contractile factors can counteract the relaxing effect of high extracellular Ca(2+) concentrations.     

Acidosis-induced protein tyrosine phosphorylation depends on Ca2+ influx via voltage-dependent Ca2+ channels in SHR aorta

The contractile response to acidosis in isolated aorta from spontaneously hypertensive rat (SHR) depends upon tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI3-kinase) and Ca2+ influx via voltage-dependent Ca2+ channels (VDCC). In this study, verapamil, a VDCC inhibitor, was shown to markedly inhibit acidic pH-induced contraction, whereas the residual contraction in the presence of verapamil was unaffected by the PI3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY-294002). Interestingly, the LY-294002-insensitive component of contraction was further inhibited by verapamil in the presence of LY-294002. Western blotting revealed that acidosis stimulated tyrosine phosphorylation of p85, which was abolished when tissues were pretreated with tyrphostin 23, a tyrosine kinase inhibitor, verapamil or EGTA. In fura-2-loaded aortic strips, acidosis induced a rise in intracellular Ca2+ ([Ca2+]i) that was partially inhibited by LY-294002. The residual increase in [Ca2+]i caused by acidosis in the presence of LY-294002 was abolished by verapamil. These findings suggest that acidosis-induced Ca2+ influx through VDCC is the upstream event leading to the tyrosine phosphorylation of PI3-kinase, which in turn contributes to the enhancement of Ca2+ entry to some extent in SHR aorta.     

Cardiovascular effects of substituted tetrahydroisoquinolines in rats.

1. A series of substituted tetrahydroisoquinolins derived from the cleavage products of tetrandrine were found to inhibit [3H]-nitrendipine binding to rat cerebral cortical membranes. Those compounds which displaced [3H]-nitrendipine binding were also able to inhibit high KCl-induced contraction of rat aorta in vitro. 2. There was a significant correlation between the ability of these tetrahydroisoquinolines to inhibit [3H]-nitrendipine binding and KCl-induced contraction (r = 0.99, P less than 0.001). 3. CPU-23 (1-(1-[(6-methoxy)-naphth-2-yl])-propyl-2-(1-piperidine)-acetyl- 6,7- dimethoxy-1,2,3,4-tetrahydroisoquinoline), one of the most potent compounds identified in this series, behaved as a simple competitive inhibitor at the [3H]-nitrendipine binding site and reduced the apparent affinity but not the maximal number of binding sites in saturation analysis. 4. In contrast to nifedipine which caused hypotension and tachycardia, CPU-23 induced both hypotension and bradycardia in a dose-dependent manner in pentobarbitone-anaesthetized Sprague-Dawley rats, spontaneously hypertensive and age-matched normotensive WKY rats. 5. It is suggested that CPU-23 may exert its cardiovascular effects via interaction with the dihydropyridine binding site on the L-type calcium channel.     

Interactions of magnesium and chloride ions on tone and contractility of vascular muscle.

Replacement of extracellular chloride ions ([Cl-]o) by other anions, on contractility and the effects of extracellular magnesium ions ([Mg2+]o) on spontaneous mechanical activity, as well as on agonist-induced responses of rat aorta and portal vein, were studied. Replacement of [Cl-]o with acetate (Ac-) or isethionate (Ise-) ions resulted in an increase and decrease, respectively, of the spontaneous mechanical activity frequency in portal vein; the amplitudes of the spontaneous mechanical activity were attenuated by Ac- and Ise- substitution. Withdrawal of [Mg2+]o in Cl(-)-containing media resulted in elevation of tension development in rat aortas, whereas a similar maneuver in media with Ac- or Ise-, substituted for [Cl-]o, resulted in abrogation of this tension development. Use of disulfonic stilbene anion-channel blockers, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate, 400-600 microM) and SITS (4,4'-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid, 400-600 microM), failed to influence either spontaneous mechanical activity or basal tone of rat portal portal vein or aortas. Incubation of DIDS or SITS in Mg(2+)-free media also failed to influence mechanical responses to withdrawal of [Mg2+]o. Use of the Cl- cation transport inhibitor bumetanide (30-80 microM) also failed to alter spontaneous mechanical activity or basal tone in either the presence or absence of [Mg2+]o. Ac- and Ise- substitution attenuated norepinephrine- and K(+)-induced contractile responses in portal vein and aorta, Caffeine-induced contractions of aortas were potentiated by withdrawal of [Mg2+]o in Cl(-)-containing media but inhibited in Ac(-)- or Ise(-)-substituted solutions. In the presence of [Mg2+]o, substitution of foreign anions resulted in alterations in the agonist contractile dose-response curves; EC50s were increased whereas maximum tensions were depressed. Withdrawal of [Mg2+]o amplified these effects. Substitution of Ac- or Ise- for [Cl-]o in the presence or absence of [Mg2+]o depressed contractions induced by Ca acetate in aortas and portal vein. These results suggest that: (1) Cl- plays an important role in regulating spontaneous mechanical activity, basal tone, and contractility in rat aorta and portal vein; and (2) Cl- probably physiologically mediates some of the effects and actions of [Mg2+]o on intracellular release of and influx of Ca2+ in these smooth muscles.     

Frequency-force relationship in guinea-pig ventricular myocardium as influenced by magnesium

Summary o li]1.|In guinea-pig papillary muscle, the characteristic relation between force of contraction and frequency is changed by the withdrawal of magnesium from the incubation medium. In magnesium-free solution, reduction of contraction frequency below 0.1 Hz leads to an increase in force of contraction which reaches its maximum at a frequency of 0.00166 Hz (i.e., one contraction every 10 min).After magnesium withdrawal, the frequency-force relationship in guinea-pig ventricular muscle resembles that of guinea-pig atrial muscle in magnesium-containing solution. li]2.|The increase by magnesium withdrawal in contractile force of guinea-pig papillary muscles contracting at low frequencies is the result of an increase in contraction velocity. The time to peak force is shortened and the relaxation time is prolonged. li]3.|After obtaining steady-state values of contractile force at 1 Hz contraction frequency, stimulation was terminated and the time course of changes in the inherent contractile activity of the muscle was determined by eliciting single contractions at time intervals of between 0.5 and 10 min duration.After cessation of stimulation, the contractile activity declines exponentially in solution containing 1.2 mM Mg²⁺; in magnesium-free solution an initial decline is followed by a slowly developing increase. This rise in contractile activity is reduced at 3.2 mM Ca²⁺ by the presence of 0.075 mM Mg²⁺ and is prevented by 0.3 mM Mg²⁺. li]4.|The increase in contractile activity obtained in the papillary muscle during rest by magnesium withdrawal depends in its magnitude on [Ca²⁺]₀. li]5.|The rested-state contractile activity of ventricular muscle in magnesium-free solution is reduced by 75% with the first and by 90% with the second contraction after onset of 1 Hz stimulation. li]6.|Possible mechanisms are discussed by which Mg²⁺ inhibits the development of rested-state contractile activity in the guinea-pig ventricular myocardium.     

Topiramate modulates pH of hippocampal CA3 neurons by combined effects on carbonic anhydrase and Cl-/HCO3- exchange

Topiramate (TPM) is an anticonvulsant whose impact on firing activity and intracellular pH (pHi) regulation of CA3 neurons was investigated. Using the 4-aminopyridine-treated hippocampal slice model bathed in bicarbonate-buffered solution, TPM (25-50 microm) reduced the frequency of epileptiform bursts and action potentials without affecting membrane potential or input resistance. Inhibitory effects of TPM were reversed by trimethylamine-induced alkalinization. TPM also lowered the steady-state pHi of BCECF-AM-loaded neuronal somata by 0.18+/-0.07 pH units in CO(2)/HCO(3)(-)-buffered solution. Subsequent to an ammonium prepulse, TPM reduced the acidotic peak but clearly slowed pHi recovery. These complex changes were mimicked by the protein phosphatase inhibitor okadaic acid. Alkalosis upon withdrawal of extracellular Cl(-) was augmented by TPM. Furthermore, at decreased pHi due to the absence of extracellular Na(+), TPM reversibly increased pHi. These findings demonstrate that TPM modulates Na(+)-independent Cl(-)/HCO(3)(-) exchange. In the nominal absence of extracellular CO(2)/HCO(3)(-) buffer, both steady-state pHi and firing of epileptiform bursts remained unchanged upon adding TPM. However, pHi recovery subsequent to an ammonium prepulse was slightly increased, as was the case in the presence of the carbonic anhydrase (CA) inhibitor acetazolamide. Thus, a slight reduction of intracellular buffer capacity by TPM may be due to an inhibitory effect on intracellular CA. Together, these findings show that TPM lowers neuronal pHi most likely due to a combined effect on Na(+)-independent Cl(-)/HCO(3)(-) exchange and CA. The apparent decrease of steady-state pHi may contribute to the anticonvulsive property of TPM.     

Effect of Hoe 694, a novel Na(+)-H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte.

1. Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride) is a Na+/H+ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pHi-regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE-1, as suggested from molecular biology studies of this protein. 2. pHi was recorded by fluorescence microscopy with carboxy SNARF-1, AM-loaded into single ventricular myocytes of guinea-pig. 3. In nominally HCO3-free media, recovery of pHi from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 microM respectively, giving the potency series: Hoe 694 congruent to DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na+) match those determined previously for cloned NHE-1 expressed in mutant fibroblasts. In the absence of extracellular Na+ (to inhibit NHE), Hoe 694 had no effect on pHi. 4. In 5% CO2/HCO3(-)-buffered solution containing DMA, pHi recovery from acidosis is mediated by Na(+)-HCO3- symport and was unaffected by Hoe 694. The drug also had no effect on pHi recovery from an alkali-load, a process largely mediated by Cl(-)-HCO3- exchange. Finally, the fall of pHi upon adding extracellular Na-lactate is assisted by H(+)-lactate symport, and this too was unaffected by Hoe 694. 5. We conclude (i) Hoe 694 has no detectable inhibitory potency for pH-regulating carriers in heart other than NHE. (ii) native NHE functioning during pHi-regulation in the cardiomyocyte is the NHE-1 isoform. These data strengthen the case for NHE-1 being the receptor for mediating the cardioprotective effects of Hoe 694.     

Characterization in rat aorta of the binding sites responsible for blockade of noradrenaline-evoked calcium entry by nisoldipine.

1. The effectiveness of the calcium antagonist, 1,4-dihydropyridine nisoldipine, as an inhibitor of contraction and 45Ca entry evoked by noradrenaline in rat aorta has been investigated and correlated with binding characteristics in intact artery. 2. Contractions evoked by noradrenaline were concentration-dependently depressed by nisoldipine (0.3-300 nM). About 60% of the response was resistant to inhibition, while KCl-induced contractions could be completely blocked. Noradrenaline-induced contractions were also less sensitive to nisoldipine inhibition than were KCl-induced contractions. 3. Preincubation of the aorta with nisoldipine in high KCl depolarizing solution increased the inhibition of the contraction evoked by a short application of noradrenaline or KCl to a similar extent. 4. The inhibition by nisoldipine of 45Ca influx evoked either by KCl depolarizing solution or by noradrenaline correlated well with the inhibition of the contractile responses. However, while KCl-stimulated 45Ca influx was totally abolished by nisoldipine (300 nM), 38% of the noradrenaline-stimulated 45Ca influx was resistant to inhibition by nisoldipine (300 nM). 5. The study of [3H]-(+)-PN 200-10 ([3H]-(+)-isradipine) binding in intact aorta showed the presence of a homogeneous population of specific binding sites. KD values were dependent on the KCl concentration in the bath while Bmax was unaffected. Binding of [3H]-(+)-isradipine was also increased in tissue exposed to noradrenaline; in the presence of 10(-5) M noradrenaline, binding parameters of [3H]-(+)-isradipine were close to the values obtained in aorta bathed in 20 mM KCl solution. 6. Displacement of [3H]-(+)-isradipine specific binding by nisoldipine was determined in segments of mesenteric artery and of aorta. The potency of nisoldipine was dependent on the incubation conditions applied to the vessel, as follows: KCl (100 mM) depolarizing solution greater than noradrenaline (10(-5) M) = KCl (25 mM) solution greater than physiological solution. The Ki value measured in aorta exposed to noradrenaline (10(-5) M) was close to the IC50 value of nisoldipine on the noradrenaline-evoked contraction. 7. The membrane potential value of rat aorta was estimated by the distribution of [3H]-tetraphenylphosphonium bromide ([3H]-TPP+), [3H]-TPP+ uptake concentration-dependently decreased when the KCl concentration in the bath was increased from 5.9 to 130 mM. Noradrenaline also concentration-dependently decreased [3H]-TPP+ uptake; the maximum effect (1-10 microns noradrenaline) was comparable in amplitude to the effect of 25 mM KCl solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium antagonism by KB-2796, a new diphenylpiperazine analogue, in dog vascular smooth muscle.

The effects of KB-2796, a new diphenylpiperazine analogue, on [3H]nitrendipine ([3H]NTD) binding, KCl-induced contraction and 45Ca influx has been examined in dog vascular smooth muscle, and compared with those of other diphenylpiperazines. In the binding study, [3H]NTD was found to bind with a high affinity to a single class of sites on aortic membranes (Kd = 0.41 nM and Bmax = 31 fmol (mg protein)-1). KB-2796 inhibited specific [3H]NTD binding in a concentration-dependent manner, with a Ki value of 0.34 microM. The other diphenylpiperazine derivatives such as flunarizine and cinnarizine also inhibited binding in the same manner. Also, in the contraction study, all the diphenylpiperazines antagonized the 50 mM KCl-induced contraction of isolated mesenteric arteries concentration-dependently. The IC50 values of the compounds for KCl-induced contraction correlated strongly with the respective Ki values obtained in the [3H]NTD binding study. In the 45Ca influx study, KB-2796 also effectively inhibited KCl-induced 45Ca influx in mesenteric arteries, with an IC50 value of 0.14 microM. This was close to the IC50 value found in the KCl-induced contraction study. These findings suggest that calcium antagonism by KB-2796 is responsible for its vasorelaxing action in vascular smooth muscle.