Normal Physiological Salt Solution Research Articles

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The effect of extracellular calcium on isolated uterine muscle contractions stimulated by acetone leaf extract of Anogeissus leiocarpa was investigated in a rat. About 12 m segment of the uterine muscle strip was mounted initially in a thermostatically regulated organ bath (37°C) containing normal Physiological Salt Solution (PSS; De Jalon) and later inphysiological salt solution devoid of CaCl2. The extract contracted the uterine muscle preparation in a concentration-related manner in normal PSS, with 0.53 mg/ml as the lowest active concentration. This contractile response was abolished by the calcium channel blocker, verapamil HCl (2 µg/ml). The extract however, did not evoke any contractile activity on the isolated tissue in a calcium-free media (PSS), even when caffeine (2 mmol) known to release calcium via the calcium induced-calcium release (CICR) mechanism was added to the perfusate. The results demonstrated the requirement for extracellular calcium for the extract mediated contractions and the inability of the extract to access calcium from intracellular storage sites such as the sarcoplasmic reticulum and the mitochondria. Therefore, the influx of Ca2+ into the cell cytosol is a pre-requisite for the extract-mediated uterine muscle contraction. Key word: Anogeissus leiocarpa, thermostatically organ bath, verapamil, caffeine, uterus.

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

Effects of GLP‐1 receptor agonist on Ca2+ handling in coronary smooth muscle cells from metabolic syndrome Ossabaw swine with coronary artery disease

Chronic effects of glucagon‐like peptide (GLP‐1) receptor agonists were studied in Ossabaw swine with metabolic syndrome (MetS). MetS was induced by hypercaloric atherogenic diet for 6 months. Subcutaneous injections were given twice daily for 6 months after induction of MetS. Consistent with clinical effects, food consumption and weight gain were attenuated. MetS increases coronary artery disease (CAD) and coronary smooth muscle (CSM) Ca2+ dysfunction. CSM cells were isolated enzymatically from the coronary arteries and Ca2+ was digitally imaged with fluorescent fura‐2. In normal physiological salt solution we released sarcoplasmic reticulum (SR) stores with caffeine. Treated cells showed a larger Ca2+ transient, ΔF360/380=0.26±0.02, compared to placebo cells, ΔF360/380=0.16±0.01 (p<0.001). In lean CSM, acute exposure to exenatide markedly increased activity of the sarcoplasmic‐endoplasmic reticulum Ca2+ ATPase (SERCA). This activity was confirmed by cyclopizaonic acid block. The increased SERCA activity may explain increased SR Ca2+ store in chronic MetS CSM. Acute exposure of placebo MetS CSM to exenatide has shown no effect on SERCA activity (n=43). Evidence for myocardial resistance to GLP‐1 in MetS is consistent with the lack of acute response. Our interpretation is that chronic exposure attenuates the decline in CSM Ca2+ handling associated with more severe CAD and dedifferentiation of CSM.(Support: NIH HL062552, TL1 TR000162, Amylin Pharmaceuticals Inc.)

The significance of extracellular Ca2+ in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M3 cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K+, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca2+ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca2+-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.

Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat.

(1) The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200-500 microm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. (2) Following incubation of IPA with 100 nm thapsigargin (Thg) in Ca2+-free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mm Ca2+, which was unaffected by either diltiazem (10 microm) or the reverse mode Na+/Ca2+ antiport inhibitor KB-R7943 (10 microm). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. (3) The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3-loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+-free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery. (4) 100 nm Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of approximately 30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+-dependent K+ and Cl- currents were blocked; it was greatly inhibited by 1 microm La3+, 1 microm Gd3+, and the IP3 receptor antagonist 2-APB (75 microm), and by replacement of extracellular cations by NMDG+. (5) In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+- and Gd3+-sensitive current, and an increased Mn2+ quench of Fura PE-3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

An important role for the Na+-Ca2+ exchanger in the decrease in cytosolic Ca2+ concentration induced by isoprenaline in the porcine coronary artery.

The role of the Na+-Ca2+ exchanger (NCX) in the mechanism of the isoprenaline (Iso)-induced vasorelaxation was investigated by simultaneously monitoring the intracellular Ca2+ concentration ([Ca2+]i) and tension of fura-2-loaded medial strips of porcine coronary arteries. Normal physiological salt solution (PSS) contained 137.3 mM Na+ and 5.9 mM K+. During the sustained phase of contraction, Iso induced only a transient decrease in [Ca2+]i when contraction was induced by depolarization with 118 mM K+ solution containing 25.2 mM Na+. When contraction was induced with 30 mM K+ in PSS containing 113.2 mM Na+, Iso induced a sustained decrease in [Ca2+]i, whereas in contractions induced by 30 mM K+ in a low Na+ (25.2 mM Na+) PSS, Iso transiently decreased [Ca2+]i. Replacement of Ca2+ with Ba2+ (which cannot be extruded by the Ca2+ pumps but can be extruded through the NCX) resulted in decreased [Ba2+]i induced by Iso in normal but not in low Na+ PSS. On the other hand, Iso induced a sustained decrease in [Ca2+]i when strips were pre-contracted by U46619, a thromboxane A2 analogue, in PSS. Various types of K+ channel blockers (iberiotoxin, 4-aminopyridine, apamin or glibenclamide) or combinations of these blockers failed to completely inhibit the Iso-induced decreases in [Ca2+]i and tension. However, Iso-induced sustained decreases in [Ca2+]i during the contraction induced by U46619 were greatly inhibited in a low Na+ PSS. The Iso-induced decrease in tension during contraction by U46619 was greatly inhibited by 2',4'-dichlorobenzamil, a forward- and reverse-mode NCX inhibitor, but not by ouabain, a selective inhibitor of Na+,K+-ATPase. These results indicate that the NCX is involved in the Iso-induced reduction of [Ca2+]i and tension of the porcine coronary arterial smooth muscle.

Role of EDHF in the vasodilatory effect of loop diuretics in guinea-pig mesenteric resistance arteries.

1. Relaxing effect of loop diuretics, piretanide and furosemide in comparison with acetylcholine (ACh) was investigated in guinea-pig isolated mesenteric resistance arteries. 2. Concentration-response curves to ACh (0.001 - 10 microM) and diuretics (0.0001 - 1 microM) were constructed in noradrenaline (10 - 30 microM)-precontracted arteries incubated either in normal physiological salt solution (PSS) or in 30 mM KCl PSS (K-PSS). 3. In PSS, maximal relaxations (R(max)) and pD(2) to ACh were 87+/-2% and 7.1+/-0.1 (n=10). L-N(G)-nitro-arginine methyl ester (L-NAME, 100 microM) reduced R(max) by 20% (P<0.01, n=7) and pD(2) by 10% (P<0.01). In contrast, indomethacin (10 microM) increased R(max) by 19% (P<0.01, n=8) and pD(2) by 10% (P<0.05). Combination of L-NAME+indomethacin reversed the effect observed with either of these inhibitors used alone. In K-PSS, R(max) was attenuated by 40% (P<0.001, n=6) compared to PSS. L-NAME reduced R(max) by 65% (P<0.01, n=5) and increased pD(2) by 15 fold. L-NAME+indomethacin suppressed the resistant relaxation. 4. In PSS+L-NAME+indomethacin, inhibitors of small (SK(Ca); apamin, 0.1 microM) and large (BK(Ca); iberiotoxin and charybdotoxin, 0.1 microM) conductance Ca(2+)-sensitive K(-)-channels used alone had little effect on the ACh-response. Combination of apamin+iberiotoxin reduced R(max) by 40% (P<0.05, n=7) while apamin+charybdotoxin fully abolished the resistant relaxation. 5. In PSS, piretanide and furosemide induced relaxation with R(max): 89+/-3% vs 84+/-5% and pD(2): 8.5+/-0.1 vs 7.7+/-0.2 (P<0.01) for piretanide (n=11) and furosemide (n=10), respectively. Endothelial abrasion suppressed relaxation to diuretics. L-NAME and indomethacin used alone or in combination did not significantly modify the response to diuretics. 6. In K-PSS, piretanide-induced relaxation was abolished whereas that to furosemide was reduced by 70% (P<0.001, n=9) compared to PSS and was suppressed by L-NAME+indomethacin. In PSS+L-NAME+indomethacin, apamin slightly reduced relaxation to diuretics whereas charybdotoxin or iberiotoxin abolished the response. 7. These results indicate that ACh-evoked relaxation is mediated by both NO/PGl(2)-dependent and -independent mechanisms. The EDHF-dependent component relies on activation of Ca(2+)-activated K(+) channels, is sensitive to a combination of apamin+charybdotoxin and to a smaller degree to a combination of apamin+iberiotoxin. Loop diuretic-induced relaxation is endothelium-dependent, appears to be mediated by NO, PGl(2) and EDHF for furosemide and EDHF only for piretanide. For the two diuretics, opening of BK(Ca) channels may be involved in the relaxation.

Source of calcium for contractile responses of large and small human intramyometrial arteries.

The role of calcium (Ca(2+)) released from intracellular stores and the entry of extracellular Ca(2+) for vasopressin (AVP)-induced responses in large and small, human, intramyometrial arteries was investigated. There was no statistical difference as revealed by pD(2) values (-log EC(50)), in the sensitivity of large and small vessels to AVP. Nimodipine caused an inhibition of contractions induced by low concentrations (10(-10) mol/l) of AVP in both types of vessels but, at higher concentration (>10(-10) mol/l), whereas responses in small arteries were diminished, in large arteries they remained unchanged. In Ca(2+)-free solution, responses of large and small arteries to potassium and to 10(-10) mol/l AVP were abolished. With 10(-6) mol/l AVP, response in small arteries was completely inhibited, whereas in large arteries it was reduced by approximately 50%. Additional experiments were done on large arteries. Thapsigargin (TSG), which causes depletion of internal Ca(2+) stores, caused a significant reduction in responses. Following treatment with TSG, responses to AVP in Ca(2+)-free solution were almost completely inhibited but arteries responded again when incubated in normal physiological salt solution. The results indicate that in contrast to large arteries, small arteries are highly dependent on extracellular Ca(2+). Response of large arteries showed considerable dependence on Ca(2+) stored internally particularly, for maximum activation.

1-Ethyl-2-benzimidazolinone stimulates endothelial K Ca channels and nitric oxide formation in rat mesenteric vessels

Hyperpolarization of most blood vessels occurs by the opening of K Ca channels. 1-Ethyl-2-benzimidazolinone (1-EBIO) is a direct activator of K Ca channels in epithelial cells and is potentially valuable for studying cellular hyperpolarization. This study reports the effects of 1-EBIO on isolated rat mesenteric beds perfused with normal (4.7 mM), or high (20 or 80 mM) K + physiological salt solution (PSS) and constricted with an α 1-adrenoceptor agonist, cirazoline (0.3–1 μM). Arterial perfusion pressures were decreased by 1-EBIO (0.1–30 nmol) in a dose- and endothelium-dependent manner. Infusion of penitrem A (100 nM), a maxi-K + channel blocker, or apamin (0.5 μM), a small-conductance (SK Ca) K + channel blocker, produced significant increases in cirazoline-mediated tone (mm Hg): 103.3±8.7 (control) vs. 156.3±14.3 (penitrem A); or 93.0±15.8 (control) vs. 114.0±15.4 (apamin). 1-EBIO relaxations were attenuated by penitrem A, while apamin, dendrotoxin (50 nM; a K V channel antagonist), or ouabain (100 μM; a sodium pump blocker) failed to alter the responses. 1-EBIO-mediated relaxations decreased significantly with increasing extracellular [K +]: relaxations to 30 nmol were 89.3%±3.2% (4.7 mM K +, normal PSS) vs. 59.5%±3.4% and 19.0%±3.9% for 20 and 80 mM K + PSS, respectively. Nω-nitro- l-arginine-methyl ester ( l-NAME; 100 μM), and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; 10 μM), selective inhibitors of nitric oxide synthase, and nitric oxide-sensitive guanylate cyclase, respectively, abolished 1-EBIO relaxations in vessels perfused with 20 or 80 mM K + PSS. We conclude that: (1) maxi-K + and SK Ca channels are present in rat mesenteric arterial vessels and actively contribute to vascular tone, (2) vasodilator action of 1-EBIO involves the opening of endothelial maxi-K + channels and nitric oxide synthesis.

Influence of chloride ions on α1-adrenoceptor mediated contraction and Ca 2+ influx in rat caudal artery

The objective of the present investigation was to compare and contrast the effects of 8-bromoguanosine 3′:5′-cyclic monophosphate (8-Bromo-cyclic GMP), an analogue of guanosine 3′:5′-cyclic monophosphate, felodipine, a dihydropyridine Ca 2+ channel antagonist, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a putative chloride channel antagonist on α 1-adrenoceptor mediated contraction and Ca 2+ influx in rat caudal artery, in normal physiological salt solution and in chloride-free solution. Isometric contractions and 45Ca 2+ influx were measured in isolated rat caudal arterial rings. Phenylephrine induced concentration-dependent contractions were inhibited by 8-Bromo-cyclic GMP (10 μM), felodipine (10 nM) and NPPB (3.0 μM). Removal of chloride ions also impaired phenylephrine-induced contractions. In chloride-free buffer, phenylephrine-induced contractions were partially inhibited by the presence of 8-Bromo-cGMP or felodipine, while NPPB had no effect. Phenylephrine induced 45Ca 2+ influx was inhibited by the presence of 8-Bromo-cyclic GMP, felodipine and NPPB. Moreover, removal of chloride ions also inhibited phenylephrine-induced 45Ca 2+ influx. The results of our study demonstrate that in the rat caudal artery the inhibitory effects of 8-Bromo-cyclic GMP, felodipine and NPPB, are mediated through a reduction of Ca 2+ influx. In addition, chloride ions, in part, play a role in α 1-adrenoceptor-mediated Ca 2+ influx. However, the influence of removal of chloride ions on phenylephrine stimulated contraction is limited. Moreover, 8-Bromo-cyclic GMP and felodipine, but not NPBB, impair phenylephrine-induced contractions in the absence of chloride ions.

Effects of pH on contraction and Ca2+ mobilization in vascular smooth muscles of the rabbit basilar artery.

The present study was undertaken to investigate the effects of extracellular pH (pHe) and intracellular pH (pHi) on 5-hydroxytryptamine (5-HT)-induced contraction and Ca2+ mobilization in vascular smooth muscles. Strip preparations of the rabbit basilar artery without endothelium were loaded with 40 microM fura-2-AM and 2 microM BCECF-AM and mounted in an organ bath. The isometric tension was recorded by using a force displacement transducer. Administration of 5-HT caused dose-dependent contraction in the rabbit basilar arteries. Acidification of pHe from 7.40 to 6.90 reduced the 5-HT-induced contraction and [Ca2+]i transients. Alkalinization of pHe from 7.40 to 7.90, on the other hand, enhanced the contraction and elevation of [Ca2+]i. In the other series of experiments, pHi (7.12 in normal PSS) was selectively altered by adding either butyric acid or trimethylamine. Intracellular acidification (pHi = 6.89) and alkalinization (pHi = 7.35) without changes in pHe produced qualitatively similar effects to those caused by extracellular acidification and alkalinization, respectively. Ca-sensitivity, which is defined as Deltatension/Delta[Ca2+]i, was not affected by the alteration of pHe nor pHi. In the Ca2+-free solution, the addition of 5-HT produced transient increases in [Ca2+]i and isometric tension that were much smaller than those in the normal physiological salt solution. The 5-HT-induced responses of [Ca2+]i and tension in the Ca2+-free solution were not affected by acidification nor alkalinization. These results suggest that a 5-HT-induced contraction is significantly modulated by pH through changing the [Ca2+]i transients, and that the change of pHi plays, at least in part, a role in the alteration of 5-HT-induced contraction resulting from acidosis or alkalosis in the rabbit basilar artery.

Contractile properties of ureters from rats with infravesical urinary outlet obstruction.

Mechanical properties of ureters from rats with infravesical urinary outflow obstruction were studied in vitro. Urinary outflow obstruction was created by partial ligation of the urethra in female rats. After 10 days a marked hypertrophy of the urinary bladder and a dilatation of the ureters were observed. Proximal and distal segments of the ureters from these animals were isolated and mounted in a wire myograph for force registration. Comparisons were made with ureters from control rats. The ureters from the rats with urinary outflow obstruction exhibited a large increase in lumen diameter and an unchanged thickness of the muscle layer. These data suggest that the dilatation of the ureters is associated with growth of the smooth muscle in the wall. All ureter preparations were relaxed in normal physiological salt solution. When the extracellular K+ concentration was increased to 20 mM the dilated ureters became spontaneously active. At [K+] in the range 20-40 mM in the presence of noradrenaline (10(-5) M) all ureters exhibited high-frequency spontaneous contractions. The dilated ureters had a lower frequency of spontaneous contractions and a higher force. The results show a pronounced remodelling of the ureter wall following infravesical outlet obstruction. The structural changes were associated with alterations in the contraction pattern of the preparations, most probably reflecting changes in the excitation-contraction coupling of the growing cells.

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle.

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection. 2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7 +/- 0.9 nmol g-1 tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 microM), omega-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 microM), aspartate (100 microM), gamma-aminobutyric acid (100 microM) or carbachol (50 microM). 3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6 +/- 12.7 nmol g-1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100-400 microM) partially protected NO (15 microM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 microM), but not from inhibition by hydroquinone or duroquinone (both 100 microM). 4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 microM; 2 h) still exhibited release of ascorbate (2.5 +/- 0.4 nmol g-1 tissue) and urate (22.2 +/- 2.9 nmol g-1 tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K+ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles. 5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms

This study was designed to test the hypothesis that 8-Br-cAMP and 8-Br-cGMP dependent relaxation of phorbol dibutyrate stimulated contractions of intact rat aorta are independent of changes in the level of myosin light chain phosphorylation. Phorbol dibutyrate stimulated contraction with a concomitant increase in myosin light chain phosphorylation in normal tissues and without an increase in myosin light chain phosphorylation in calcium-depleted tissues. Phorbol dibutyrate stimulated contractions in normal CaCl2-containing physiological salt solution were relaxed in a concentration-dependent manner by 8-Br-cAMP and 8-Br-cGMP. Phorbol dibutyrate-induced contractions in the absence of Ca2+ were only relaxed by 8-Br-cGMP; 8-Br-cAMP had no effect The relaxation induced by 8-Br-cGMP was associated with a decrease in myosin light chain phosphorylation suggesting that cGMP-dependent protein kinase may alter the activity of either the myosin light chain kinase or phosphatase. The relaxation induced by 8-Br-cAMP was not associated with a decrease in phosphorylation suggesting that cAMP-dependent protein kinase may uncouple myosin light chain phosphorylation from force.

CPA induces a sustained increase in [Ca2+]i of endothelial cells in situ and relaxes porcine coronary artery.

Using fura 2 fluorometry, we investigated the effect of cyclopiazonic acid (CPA), an inhibitor of Ca2+ pump adenosinetriphosphatase of the endoplasmic reticulum, on cystosolic Ca2+ concentration ([Ca2+]i) and tension in porcine aortic valvular endothelial cells and coronary arterial strips with endothelium. In normal physiological salt solution, CPA induced a sustained increase in [Ca2+]i in the valvular strips, whereas in Ca(2+)-free physiological salt solution, CPA elicited a transient elevation of [Ca2+]i. CPA(30 microM) relaxed coronary strips with endothelium precontracted by 100 nM U-46619; this relaxation was partially inhibited by N omega-nitro-L-arginine (100 microM). These results indicate that the CPA-induced increase in [Ca2+]i depends on the Ca2+ release and the Ca2+ influx in the endothelial cells in situ and that the CPA-induced endothelium-dependent decreases in [Ca2+]i and tension in the smooth muscle are due to the combined effect of N omega-nitro-L-arginine-sensitive and -resistant factors.

Effects of diltiazem and its analogue siratiazem on contraction and 45Ca 2+ uptake in sheep coronary artery rings

1. 1. Siratiazem, an analogue of diltiazem designed to be resistant to N-demethylation, was compared with diltiazem for inhibition of Ca 2+-induced contraction of depolarized coronary artery rings from the sheep. There was no significant difference in potency between siratiazem and diltiazem in the presence of normal physiological salt solution (IC 25 for siratiazem 0.13 ± 0.04 μM and for diltiazem 0.08 ± 0.02 μM) or one mimicking some of the conditions that occur during myocardial ischaemia (hypoxia, acidosis, reduced glucose and addition of lactate). 2. 2. K +-stimulated 45Ca 2+ uptake in coronary artery rings was also inhibited by siratiazem and diltiazem with similar potencies. 3. 3. It is concluded that siratiazem inhibits Ca 2+ entry in coronary vascular smooth muscle equipotently with diltiazem and that this effect is not modified by some of the changes that occur during myocardial ischaemia.

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification

1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

Role of GTP-protein and endothelium in contraction induced by ethanol in pig coronary artery.

1. We examined the effects of ethanol on the contractility of strips of porcine coronary artery, with and without endothelium, and following permeabilization with alpha-toxin, and of aortic valvular endothelial cells, in situ. Changes in cytosolic Ca2+ concentration ([Ca2+]i) of the coronary artery smooth muscle cells and of the valvular endothelial cells were monitored using front-surface fluorometry of the calcium indicator dye, fura-2. In permeabilized preparations, [Ca2+]i was clamped using 10 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid (EGTA) and 10 microM A23187 (a calcium ionophore). 2. The strips without endothelium were placed in normal physiological salt solution (normal PSS) in the presence of ethanol (100-1000 mM). There were dose-dependent increases in [Ca2+]i and a rapid sustained rise in tension. In Ca(2+)-free PSS, ethanol increased [Ca2+]i and tension, similar to, but much smaller than, findings with normal PSS. 3. For a given change in [Ca2+]i induced by ethanol, the developed tension was greater than that observed during contractions induced by high [K+]o. Thus, the [Ca2+]-tension curve for ethanol was shifted to the left of that for high [K+]o. The [Ca2+]-tension curve for the contraction induced by ethanol in the absence of extracellular Ca2+ was shifted further to the left from that obtained in the presence of [Ca2+]o. 4. The mechanisms involved in this Ca(2+)-sensitizing effect of ethanol were investigated using alpha-toxin-permeabilized coronary medial strips. Ethanol increased the tension development, in a concentration-dependent manner, at a fixed concentration of Ca2+ (pCa = 6.3) in the presence of guanosine-5'-triphosphate (GTP), an effect antagonized by guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S), a non-hydrolysable GDP analogue. 5. With intact endothelium, the ethanol-induced tension development was markedly reduced, although inhibition in the increase in [Ca2+]i was slight. The [Ca2+]-tension relationship of this contraction overlapped with that obtained with high [K+]o-induced contraction and was shifted to the right from that obtained in the absence of the endothelium. This endothelium-dependent reduction of [Ca2+]i and tension induced by ethanol was inhibited when the strips were exposed to NG-monomethyl-L-arginine (L-NMMA). 6. Ethanol induced a gradual and sustained increase in [Ca2+]i in normal PSS, and a transient, concentration-dependent increase in [Ca2+]i in Ca(2+)-free PSS in porcine aortic valvular endothelial cells in situ.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of rubidium on responses to potassium channel openers in rat isolated aorta.

1. In a physiological salt solution (PSS) in which potassium (K) was replaced by rubidium (Rb), segments of rat aorta precontracted with 20 mM RbCl were fully relaxed by K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were inhibited by glibenclamide. 2. Segments of rat aorta bathed in normal PSS and precontracted with 20 mM KCl were also relaxed by these K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were glibenclamide-sensitive. However, the absolute potencies of the K-channel openers were approximately four times greater in normal PSS than in RbPSS. 3. In RbPSS, minoxidil sulphate relaxed segments of aorta precontracted with 20 mM RbCl by approximately 20% whereas in normal PSS it fully relaxed those contracted with 20 mM KCl. 4. In RbPSS, levcromakalim-induced relaxation of aortic segments precontracted with 20 mM RbCl was initially well-maintained but then faded by approximately 60% of the initial relaxation to a new, stable level. Subsequent exposure to RP 49356 or to higher concentrations of levcromakalim was without further relaxant effect. Similar changes were observed when RP 49356 was the initial relaxant and tissues were exposed to either RP 49356 or levcromakalim. In normal PSS, levcromakalim- or RP 49356-induced relaxation of contractions produced by 20 mM KCl was well-maintained. 5. In RbPSS, minoxidil sulphate-induced relaxation of aortic segments precontracted with 20 mM RbCl was well-maintained. Subsequent exposure to either levcromakalim or to RP 49356 produced further tissue relaxation. 6. In RbPSS, levcromakalim produced no detectable increase in either 86Rb- or 42K-efflux from rat aortic strips. In normal PSS, a significant increase in the exchange of both isotopes was detected.7. Levcromakalim hyperpolarized segments of rat aorta bathed both in normal PSS and after depolarization by the addition of 20 mM KCI. Exposure to RbPSS depolarized the tissue and under these conditions, levcromakalim had no effect on membrane potential.8. In Rb- and normal PSS, levcromakalim produced a similar degree of inhibition of the refilling of then or adrenaline-sensitive Ca store.9. It is concluded that millimolar concentrations of Rb inhibit the plasmalemmal ATP-sensitive K-channels (KATP) which are the target of the K-channel openers. The relaxant actions of the K-channel openers in both normal and Rb-PSS and the inhibition of these effects by glibenclamide may reflect a functional interaction between these agents at ATP-binding sites associated with both KATP and with intracellular structures including Ca stores.

Extracellular Ca(2+)-dependent potentiation by cocaine of serotonin- and norepinephrine-induced contractions in rat vascular smooth muscle.

Using front-surface fluorometry, we determined the effects of cocaine on force and cytosolic Ca2+ concentration ([Ca2+]i) in the rat aorta. We also examined the effects of cocaine on 45Ca2+ influx. Cocaine (10(-7) to 10(-4) M) alone did not alter the resting level of [Ca2+]i and force. Cocaine (< 10(-4) M), in a concentration-dependent manner, potentiated the 10(-6) M serotonin (5-HT)-induced or 10(-8) M norepinephrine (NE)-induced sustained increase in [Ca2+]i and force in the presence of extracellular Ca2+, whereas it had no potentiating effects in Ca(2+)-free solution. Similar potentiating effects of cocaine were observed in pharmacologically denervated strips. Cocaine (10(-5) M) produced a leftward shift of concentration-response curves for both 5-HT- and NE-induced increases in [Ca2+]i and force with no effect on the maximal response or the relations between [Ca2+]i and force. Cocaine (10(-5) M also accelerated the 45Ca2+ influx during activation by 10(-6) M 5-HT or by 10(-8) M NE. Cocaine (> 10(-3) M) inhibited 5-HT-, NE-, and high-K+ depolarization-induced contractions accompanied by decreases in [Ca2+]i in normal physiological salt solution and 5-HT- or NE-induced transient increase in [Ca2+]i and force in Ca(2+)-free physiological salt solution. Thus, low concentrations of cocaine potentiate NE- or 5-HT-induced contraction by augmenting the increase in [Ca2+]i. These potentiating effects may derive from either an increase in the affinity of the receptors to agonists or an increase in the Ca2+ influx. On the other hand, high concentrations of cocaine (> 10(-3) M) have a relaxant effect on vascular smooth muscle, as a result of a decrease in [Ca2+]i.