Divergent pharmacological effects of three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine and calmidazolium, on isometric tension development and myosin light chain phosphorylation in intact bovine tracheal smooth muscle

To investigate the usefulness of calmodulin antagonists in intact cell systems, effects of three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine (CPZ) and calmidazolium on isometric tension development and myosin light chain (P-LC) phosphorylation in bovine tracheal smooth muscle strips were compared to inhibition of purified myosin light chain kinase activity. These antagonists inhibited the Ca++-calmodulin-induced activation of myosin light chain kinase in a concentration-dependent manner, with IC50 values of 1.0 (calmidazolium), 25 (W-7) and 65 microM (CPZ), respectively. Inhibitory effects of these antagonists were abolished with increasing concentrations of calmodulin. However, when these antagonists were used in intact smooth muscle strips, the gradation of potencies did not parallel the anticalmodulin activities. W-7 (100 microM) exhibited a similar extent of antagonism between the contractile responses to carbachol and KCl. The increase in P-LC phosphate content in response to 1-min stimulation with 10(-5) M carbachol was inhibited by W-7. CPZ exhibited an unexpectedly potent antagonism on carbachol-induced isometric tension development and P-LC phosphorylation. Atropine showed an antagonism similar to CPZ. CPZ and verapamil had similar antagonistic effects on KCI-induced contractions. Calmidazolium (50 microM) produced no significant inhibition on carbachol-induced isometric tension development and P-LC phosphorylation in intact smooth muscle strips. It may be concluded that 1) W-7 antagonizes the smooth muscle contraction through the inhibition of the initial increase in the P-LC phosphorylation; 2) CPZ produces effects other than calmodulin antagonism; and 3) calmidazolium is not effective in intact smooth muscle strips.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium and calmodulin antagonists binding to calmodulin and relaxation of coronary segments

Ca++ antagonist drugs (also known as Ca++ channel blockers) and the calmodulin antagonists trifluoperazine (TFP) and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] were capable of half-maximally relaxing porcine coronary segments at 1.5 X 10(-10) M for felodipine, 6.5 X 10(-8) M for verapamil, 2.6 X 10(-7) M for diltiazem, 7 X 10(-7) M for prenylamine, 7 X 10(-6) M for TFP and 45 X 10(-6) M for W-7. Their correspondent binding to calmodulin was half-maximal at 2.8 X 10(-6), 30 X 10(-6), 80 X 10(-6), 5 X 10(-7), 5.0 X 10(-6) and 11 X 10(-6) M, respectively. Only prenylamine, TFP and W-7 were capable of relaxing coronaries over the same concentration range in which they bind to calmodulin. The relaxations produced by these calmodulin antagonists and prenylamine could not be overcome by contractile agonists which release Ca++ from internal stores (histamine and serotonin), whereas the relaxations produced by felodipine, verapamil and diltiazem were readily reversed by either of these agonists. This is consistent with TFP and W-7 and to some degree prenylamine-inducing vasodilation by calmodulin antagonism and with felodipine, verapamil and diltiazem vasodilating through Ca++ antagonism of Ca++ channels at the level of the cell membrane.     

Vasodilatory action of HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], a novel calcium antagonist with no effect on cardiac function

A novel compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) was found to be a potent relaxant of blood vessels. Rabbit aortic strips contracted by 18 mM KCl relaxed in the presence of HA1004, with an ED50 value of 2.2 X 10(-6) M. The isolated guinea-pig atria on right ventricular papillary muscles did not respond to HA1004, even at a concentration of 3 X 10(-4) M. HA1004, like verapamil, produced a competitive inhibition of the Ca++-induced contraction of the depolarized rabbit aorta. The pA2 value of HA1004 for the Ca++-induced contraction was 6.60. Atropine, propranolol, theophylline or indomethacin had no effect on the HA1004-induced relaxation. HA1004 (3 X 10(-6) M) inhibited the contraction produced by Ca++ ionophore, A23187, whereas verapamil or diltiazem had no effect on this contraction, even at a concentration of 3 X 10(-5) M. Moreover, HA1004 produced a competitive inhibition of Ca++-induced contraction of the A23187-treated aorta and inhibited the phenylephrine-induced contraction elicited in ca++-free solution, thereby suggesting that this drug affects intracellular rather than extracellular Ca++. The present findings indicate that HA1004 is a novel calcium antagonistic vasodilator with no effect on cardiac function and is apparently of a different class of calcium antagonist from verapamil.     

Inhibitory effects of a synthetic atrial peptide on contractions and 45Ca fluxes in vascular smooth muscle

The mechanism of a synthetic atrial peptide (APII)-induced inhibition of smooth muscle contractility was investigated by studying its effects on tension development and 45Ca fluxes in isolated rabbit aorta. APII (10(-9) to 10(-7) M) produced a dose-dependent relaxation of contractions produced by alpha adrenoceptor activation with norepinephrine (NE; 10(-6) M). APII was a potent relaxant of NE contraction with an IC50 = 1.1 X 10(-8) M, with 10(-7) M APII causing a 97% relaxation. APII also produced a dose-dependent inhibition of NE contraction when added to the resting muscle before the exposure to NE. The relaxing effects of APII were found to be endothelium independent. In contrast, APII was only marginally effective in relaxing high-K+ contraction, with 10(-7) M APII causing only 17% relaxation. Furthermore, when a NE contraction was obtained on top of a high-K+ contraction, APII was still capable of relaxing the NE component. APII was similarly more effective in inhibiting NE-stimulated 45Ca influx than high-K+-stimulated 45Ca influx, indicating selective action of APII on the receptor-operated Ca++ channels. This was in contrast to D600, a well known Ca++ antagonist, which had a more selective inhibitory effect on the potential-operated Ca++ channels. The data presented indicate that APII is a potent relaxant of contractions produced by receptor-agonists involving 45Ca influx through receptor-operated Ca++ channels. APII may also prove to be a very useful tool to further distinguish and define receptor-operated Ca++ channels and potential-operated Ca++ channels in vascular smooth muscle.     

Myosin phosphorylation-independent contraction induced by phorbol ester in vascular smooth muscle.

In isolated rat aorta, carotid artery, tail artery, rabbit aorta and mesenteric artery, but not in ear artery, 1 microM 12-deoxyphorbol 13-isobutyrate (DPB) induced a sustained contraction. However, DPB increased cytosolic Ca++ concentration ([Ca++]i) only in rat aorta and carotid artery. Similar results were obtained with phorbol 12,13-dibutyrate, although the inactive phorbol ester, 4-alpha-phorbol 12,13-dibutyrate, was ineffective. In rat aorta, DPB-induced contraction was followed by an increase in 20 kDa myosin light chain (MLC) phosphorylation. Both contraction and MLC phosphorylation stimulated by DPB were greater than those due to high K+ for a given increase in [Ca++]i. A Ca++ channel blocker, verapamil, decreased the DPB-induced increments in [Ca++]i and MLC phosphorylation to their respective resting levels, although contraction was inhibited only slightly. In the absence of external Ca++ (with 0.5 mM ethyleneglycol bis(beta-aminoethyl-ether)tetraacetic acid), DPB induced sustained contraction without increasing [Ca++]i or MLC phosphorylation. This contraction was followed by an increase in stiffness and force recovery after a shortening step. These results suggest that the contraction induced by DPB in rat aorta is due to increase in [Ca++]i followed by MLC phosphorylation and Ca++ sensitization of MLC phosphorylation. In the presence of verapamil or in the absence of external Ca++, DPB may increase cross-bridge cycling by activating an unknown mechanism that is not dependent on an increase in MLC phosphorylation.     

Calcium dependence of phorbol 12,13-dibutyrate-induced force and myosin light chain phosphorylation in arterial smooth muscle

Phorbol dibutyrate (PDB) is an activator of protein kinase C and has been observed to cause a slow developing contraction in vascular smooth muscle. The mechanism of phorbol ester-induced contraction is unknown. We studied the Ca++-dependence of, and the degree of myosin light chain phosphorylation (MLC-P), during PDB-induced contractions in rabbit aortic rings. PDB elicited concentration-dependent contractions (3 X 10(-8) to 10(-6) M) in rabbit aortic rings incubated in normal (1.6 mM Ca++) physiologic salt solution (PSS). Addition of the Ca++-channel blocker nifedipine (0.1 microM) to PSS or removal or Ca++ from PSS significantly reduced the contractile responses to PDB. Depletion of Ca++ by repeated washes in O Ca++-PSS containing 10(-3) M ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid reduced, but did not eliminate, the responses to PDB. In PSS, PDB significantly increased the fraction of phosphorylated MLC/total MLC to 0.33 from a resting value of 0.20. Ca++ depletion reduced the resting fraction (MLC-P/MLC) to 0.14. PDB-stimulated contractions in Ca++-depleted tissues occurred in the absence of significant increases in MLC-P. Sodium nitroprusside partially relaxed PDB-induced contractions by approximately 50% whether elicited in the presence of 1.6 mM Ca++ or after Ca++ depletion. In both cases relaxation occurred in the absence of statistically significant decreases in MLC phosphorylation. Ca++-dependent MLC phosphorylation may account for a component of the PDB contractile response in rabbit aorta. Studies in the absence of Ca++ suggest that PDB may activate contraction without concomitant MLC-P.     

Effects of norepinephrine on contraction and hydrolysis of phosphatidylinositols in rat aorta

The purpose of this study was to investigate the relationship between norepinephrine-induced contraction and hydrolysis of phosphatidylinositols in rat aorta. Norepinephrine-induced contraction was associated with increased accumulation of the hydrolytic products of the phosphatidylinositols, inositol monophosphate and phosphatidic acid. Norepinephrine also induced significant decreases in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The alpha-1 adrenoceptor antagonist, prazosin, exposure to the Ca++ channel modulator, nifedipine, and removal of extracellular Ca++ inhibited the accumulation of inositol monophosphate and contraction due to norepinephrine. These results suggest that the contraction induced by norepinephrine may be mediated by processes associated with hydrolysis of phosphatidylinositols. The hydrolysis may occur through Ca++-dependent activation of phospholipase C by alpha-1 adrenoceptor agonists.     

Sustained contraction of vascular smooth muscle: calcium influx or C-kinase activation?

We have investigated the relative contributions of Ca++ influx and C-kinase activation to the sustained contraction of smooth muscle of rabbit aorta. In physiological salt solution (PSS), the alpha adrenergic agonist, phenylephrine (PhE), induced a rapid initial contraction followed by a maintained tonic contraction whereas the C-kinase activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused only a slow tonic contractile response. Both PhE- and TPA-induced contractions were accompanied by a significant increase in the unidirectional 45Ca influx. The tonic phase of PhE contraction and the slow contractile response of TPA also were reduced, but not abolished completely in Ca++-free solution containing 2 mM ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid. In addition, the relatively specific C-kinase inhibitor, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], reversibly inhibited the TPA-induced contraction in PSS and almost abolished the TPA response in Ca++-free solution. On the other hand, H-7 caused only partial inhibition (30.2% +/- 4.09, n = 5) of the PhE sustained contraction in PSS and abolished completely the residual PhE maintained response in Ca++-free solution. The H-7 inhibition of the PhE sustained contraction was reversible in both PSS and Ca++-free solution. Furthermore, TPA alone could not maintain the contractile response initiated by a high K+ depolarizing solution upon replacement of the high K+ solution by normal PSS. These findings emphasize the importance of Ca++ influx and suggest only a minor role of C-kinase in maintaining the tonic contraction of vascular smooth muscle.     

Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Effects on calmodulin of bepridil, an antianginal agent

Using biopharmacological techniques, we determined the effect on calmodulin of bepridil, a Ca++ channel blocker. We used two Ca++/calmodulin-dependent enzymes, Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain and myosin light chain kinase from chicken gizzard. Bepridil inhibited the calmodulin-induced activation of Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase and the concentration of this drug producing 50% inhibition (IC50) of this enzyme was 8 microM. There was no significant effect on unactivated Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase (in the absence of Ca++-calmodulin), up to a concentration of 100 microM. Bepridil inhibited specifically Ca++/calmodulin-dependent phosphorylation of chicken gizzard myosin light chain with an IC50 value of 18 microM. Moreover, this agent produced a marked displacement of [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an antagonist that interacts selectively with calmodulin. The influence of bepridil on the dose-response curves of mesenteric arterial strips for CaCl2, norepinephrine and serotonin differed from the influence seen with nifedipine, another Ca++ blocker. Bepridil (100 microM) suppressed the Ca++-induced contraction of saponin-skinned mesenteric arteries and calmodulin (26 microM) reversed partly the relaxant effect of this agent. These results suggest that the effect of bepridil on the cardiovascular system is due not only to its Ca++ channel blocking action but also to a calmodulin antagonistic action.     

Vascular and cardiac effects of a new dihydropyridine derivative, YC-170: a comparison with Bay K 8644

The pharmacological effects of YC-170, a new dihydropyridine derivative, were studied in the rabbit aortic strips and guinea pig cardiac preparations and compared with those of Bay K 8644. In the rabbit aortic strips, YC-170 produced contraction in normal physiological saline solution ([K+]0 = 5.9 mM) in a concentration-dependent manner. Increasing the [K+]0 of the medium to 15 mM enhanced the contractile response. The maximum contraction produced by YC-170 at [K+]0 of 15 mM was comparable to that by Bay K 8644. However, YC-170 induced relaxation when the strip was contracted by 60 mM K+. In guinea pig left atrium, YC-170 produced a positive inotropic effect in a concentration-dependent manner, but its extent was far less than that of Bay K 8644. Like Bay K 8644, however, YC-170 increased the time to peak tension and relaxation time of the isometric tension, and prolonged the action potential duration. YC-170 failed to produce a positive inotropic action in the papillary muscle in which Bay K 8644 was a potent positive inotropic agent. In spontaneously beating right atria, YC-170 caused a negative chronotropic effect, whereas Bay K 8644 a positive one. The positive inotropic and vasoconstrictor effects of YC-170 were antagonized competitively by a Ca++ antagonist nicardipine. When the left atria were depolarized with high-K+ medium, the positive inotropic effect of YC-170 was attenuated progressively with increasing [K+]0 and at 13.2 mM K+ a negative inotropic effect was induced by YC-170.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of aortic calcium and contractility in male, female and lactating female rats.

Catecholamine-induced vascular smooth muscle contration is enhanced in female animals and in presence of emale sex hormones in vitro. Androgens appear to depress these responses. Sex steroids may also alter calcium ion (Ca++) binding and metabolism. We compared contractility as well as quantity and relative lability of tissue calcium pools in male and female rat isolated aortic strips bathed in Ca++-free solution. We also studied aortic strips from 21-day postpartum lactating female rats to determine the effects of previous high circulating levels of female sex steroids (present during pregnancy) and prolactin (present during lactation). Rat aortic strips were found to contain loosely and more tightly held calcium stores. Strips from males were unresponsive in Ca++-free solution unless previously exposed to a Ca++-rich bathing medium. They accumulated more tissue calcium when bathed in Ca++-rich solution than did strips from females. This extra calcium appears to reside in the loosely-held fraction. Tissues from males first incubated in Ca++-rich solution to enhance the loosely held fraction respond more readily in Ca++-free solution to a high potassium (K+) concentration than to epinephrine. Strips from females respond about equally to high K+ or epinephrine whereas aorta from lactating female rats are much more responsive to epinephrine in Ca++-free solution and gain less calcium in Ca++-rich medium than those of the other rats. These data suggest that in the presence of high circulating levels of female sex hormones or other female factors (e.g., prolactin) enhanced binding or sequestration of potential activator ions occurs which increases the responsiveness of the tissue to catecholamines. Male sex hormones and/or factors promote the capacity of the rat aorta to gain a more loosely held calcium fraction which is readily used for contraction by K+ depolarization.     

Effect of forskolin on cytosolic Ca++ level and contraction in vascular smooth muscle

The effects of forskolin, an activator of adenylate cyclase, on cytoplasmic Ca++ level ([Ca++]cyt) measured simultaneously with muscle tension using fura-2-Ca++ fluorescence were examined in isolated smooth muscle of rat aorta. Forskolin decreased muscle tension and [Ca++]cyt in resting aorta whereas both norepinephrine and high K+ solution produced sustained increase in muscle tension and [Ca++]cyt. Addition of forskolin during the sustained contractions decreased muscle tension more strongly than [Ca++]cyt. Norepinephrine-induced contraction was more sensitive to forskolin than high K+-induced contraction. The inhibitory effect of forskolin was attenuated when the concentration of norepinephrine or K+ was increased. Cumulative addition of norepinephrine or K+ induced a concentration-dependent increase in both [Ca++]cyt and muscle tension and a positive [Ca++]cyt-tension correlation was observed. In the presence of 0.1 microM forskolin, the norepinephrine-induced increments in [Ca++]cyt and muscle tension were inhibited without changing the [Ca++]cyt-tension relationship. In the presence of a higher concentration (1 microM) of forskolin, muscle tension was inhibited more strongly with only a small additional decrease in [Ca++]cyt resulting in a shift of the [Ca++]cyt-tension relationship. Norepinephrine induced transient increments in [Ca++]cyt and muscle tension in Ca++-free solution and forskolin inhibited these changes. These results suggest that forskolin has concentration-dependent inhibitory effects on vascular contractility to decrease [Ca++]cyt at lower concentrations and to decrease the sensitivity of contractile elements to Ca++ at higher concentrations.     

Mechanism of action of a novel antivasospasm drug, HA1077

HA1077, but not the potent calcium entry blocker nicardipine reversed experimental chronic cerebral vasospasm induced in a two-hemorrhage canine model. The i.a. administration of HA1077 produced significant increases in vertebral blood flow in dogs. The effects of HA1077 on the vascular responses were studied in vitro. Spiral strips of rabbit aorta were mounted for isometric tension recording in physiological salt solution. HA1077 produced a competitive inhibition of the Ca++-induced contraction of the depolarized rabbit aorta. The pA2 of HA1077 for the Ca@-induced contraction was 6.71. The inhibitory effect of HA1077 on the KCl-induced contraction was not altered by atropine, propranolol, theophylline or indomethacin. HA1077 (10(-8) to 10(-4) M) inhibited contractile responses to KCl, phenylephrine (PHE) and prostaglandin (PG) F2 alpha similarly, whereas verapamil, diltiazem and nicardipine were much less effective in blocking the contractions induced by PHE or PG. Even in Ca++-free physiological salt solution, both PHE and PG were capable of contracting the aorta. These Ca++-free contractile responses to PHE and PG were antagonized effectively by HA1077. Verapamil failed to inhibit these contractions. We also investigated the effects of HA1077 on guinea pig heart contractility. In contrast to calcium entry blockers (which are known to have a direct negative inotropic effect), HA1077 did not change the developed tension in the left atrium at concentrations up to 3 X 10(-4) M. The present evidence demonstrates that the novel antivasospasm drug HA1077 is a class of calcium antagonists different from the calcium entry blockers.     

Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh-induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13-acetate, induced MLC phosphorylation and muscle contraction in a dose- and time-dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction.     

An endothelium-dependent contraction induced by A-23187, a Ca++ ionophore in canine basilar artery

In most isolated canine basilar arteries tested, Ca++ ionophore A-23187 induced a small relaxation followed by a transient contraction. Both contraction and relaxation were abolished by removal of endothelium. The endothelium-dependent contraction induced by A-23187 was attenuated by a phospholipase A2 inhibitor (quinacrine), cyclooxygenase inhibitors (aspirin and indomethacin), a thromboxane A2 (TXA2) synthetase inhibitor (OKY-046) and a TXA2 antagonist (ONO-3708). The A-23187-induced contraction was abolished by lowering the Ca++ concentration of medium to 10%, whereas the contraction induced by 9,11-epithio-11,12-methano-TXA2 (STA2) was attenuated slightly by lowering [Ca++]. The A-23187-induced contraction was reduced markedly by nifedipine (10(-9) to 10(-7) M), but the STA2-induced contraction was only attenuated slightly by nifedipine. Bay K 8644 did not affect the A-23187- and STA2-induced contractions. The present experiments demonstrate that A-23187 induced an endothelium-dependent contraction in canine basilar artery, and suggest that Ca++ might play a key role in production of an endothelium-derived contracting factor (probably TXA2).     

Pharmacologic characterization of SK&F 104078, a novel alpha-2 adrenoceptor antagonist which discriminates between pre- and postjunctional alpha-2 adrenoceptors

SK&F 104078 has been reported to be a moderately potent antagonist at postjunctional alpha-2 adrenoceptors in canine saphenous vein, rabbit saphenous vein and canine saphenous artery (KB = 76-150 nM). In contrast, SK&F 104078 has been found to have essentially no affinity for prejunctional alpha-2 adrenoceptors in the guinea pig atrium. To characterize further the pharmacology of SK&F 104078 we have examined its effects in several additional alpha-2 adrenoceptor models and on several non alpha adrenoceptor-mediated vascular responses. SK&F 104078 does not block the neuroinhibitory effect of alpha methylnorepinephrine in the guinea pig ileum. In contrast, in the rat vas deferens, high concentrations of SK&F 104078 (3-30 microM) antagonized the neuroinhibitory effect of UK 14,304; however, the antagonism was not competitive. At concentrations up to 1 microM, SK&F 104078 did not potentiate [3H]overflow from guinea pig vas deferens or guinea pig atrium prelabeled with [3H]norepinephrine, indicating no prejunctional alpha-2 adrenoceptor blocking activity in these tissues. SK&F 104078 is a competitive antagonist at alpha-1 adrenoceptors, and at 5-hydroxytryptamine2 receptors, as demonstrated by blockade of norepinephrine- and serotonin-induced contraction in the rabbit aorta, with KB values of 155 and 20 nM, respectively. At concentrations up to 10 microM, SK&F 104078 does not depress angiotensin II-induced contraction of rabbit aorta. At 1 microM, no depression of the response to Ca++ in depolarized rabbit aorta is observed, although a significant inhibition of this response is seen at 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of thromboxane A(2)-induced Cl(-) secretion by antidiarrhea drug loperamide in isolated rat colon

The antitumor drug irinotecan clinically causes severe diarrhea as a side effect. Thromboxane A(2) (TXA(2)), released by irinotecan, has been shown to be a novel physiological stimulant of Cl(-) secretion in the rat colon. Herein, we examined the effect of loperamide, an antidiarrhea drug, on Cl(-) secretion induced by irinotecan; 9, 11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable TXA(2) analog; and prostaglandin E(2) (PGE(2)) by using isolated mucosae of the rat colon. In the presence of atropine, loperamide in a concentration-dependent manner inhibited the Cl(-) secretion induced by irinotecan, STA(2), and PGE(2). However, the drug inhibited more effectively the irinotecan- and STA(2)-induced secretion (IC(50) = 0. 7 and 1.2 microM, respectively) than the PGE(2)-induced secretion (IC(50) = 23 microM). Naloxone, an opiate antagonist, did not affect the antisecretory action of loperamide. Similar to the case for loperamide, W-7, a specific calmodulin antagonist, inhibited more effectively the STA(2)-induced Cl(-) secretion (IC(50) = 5 microM) than the PGE(2)-induced secretion (IC(50) = 36 microM). W-5, a low-affinity calmodulin antagonist (a dechlorinated control analog of W-7), also inhibited the STA(2)-induced secretion, but this effect was much less than that of W-7. STA(2)-induced increase in the intracellular free Ca(2+) concentration of single colonic crypt cells was not affected by loperamide. We suggest that loperamide efficiently inhibits the TXA(2)-induced secretion by blocking the calmodulin system in the colonic epithelium. The present results may explain why coadministration of loperamide with irinotecan is clinically efficient for avoiding the irinotecan-induced side effect of diarrhea.     

Involvement of endothelial cells in relaxation and contraction responses of the aorta to isoproterenol in naive and streptozotocin-induced diabetic rats

Experiments were designed to investigate the importance of the endothelium in the relaxation of isolated rat aorta caused by a beta adrenoceptor agonist. Mechanical removal of the endothelium attenuated the relaxation induced by isoproterenol (ISO) and did not affect the relaxation produced by forskolin and by sodium nitroprusside. High concentrations of ISO produced an increase in the resting tension of aortic strips with and without endothelium in a concentration-dependent manner. Mechanical removal of the endothelium or treatment with methylene blue enhanced the maximal contraction induced by ISO. Phentolamine antagonized the contractile responses induced by ISO. In the case of streptozotocin-induced diabetic rats, both aortic strips with and without endothelium generated concentration-response curves for ISO-induced relaxation that were shifted to the right. The relaxant responses to forskolin and sodium nitroprusside were not significantly different between vessels from diabetic and age-matched control rats. In both aortic strips with and without endothelium, the maximal contraction in response to high concentrations of ISO was significantly enhanced in strips from diabetic rats. These results suggest that ISO-induced relaxation of aortic strips with endothelium is mediated by beta adrenoceptors on both the endothelium and the smooth muscle, and high concentrations of ISO produce an increase in the resting tension through alpha adrenoceptors. It is further suggested that the decreased relaxant response of the aorta to ISO in diabetes may be due to decreased density or affinity of beta adrenoceptors on the smooth muscle.