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.     

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.     

Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes.

We have previously shown that stretching cardiac myocytes evokes activation of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and 90-kD ribosomal S6 kinase (p90rsk). To clarify the signal transduction pathways from external mechanical stress to nuclear gene expression in stretch-induced cardiac hypertrophy, we have elucidated protein kinase cascade of phosphorylation by examining the time course of activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinase (MAPKK), MAPKs, and p90rsk in neonatal rat cardiac myocytes. Mechanical stretch transiently increased the activity of MAPKKKs. An increase in MAPKKKs activity was first detected at 1 min and maximal activation was observed at 2 min after stretch. The activity of MAPKK was increased by stretch from 1-2 min, with a peak at 5 min after stretch. In addition, MAPKs and p90rsk were maximally activated at 8 min and at 10 approximately 30 min after stretch, respectively. Raf-1 kinase (Raf-1) and (MAPK/extracellular signal-regulated kinase) kinase kinase (MEKK), both of which have MAPKKK activity, were also activated by stretching cardiac myocytes for 2 min. The angiotensin II receptor antagonist partially suppressed activation of Raf-1 and MAPKs by stretch. The stretch-induced hypertrophic responses such as activation of Raf-1 and MAPKs and an increase in amino acid uptake was partially dependent on PKC, while a PKC inhibitor completely abolished MAPK activation by angiotensin II. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1 and MEKK, MAPKK, MAPKs and p90rsk, and that angiotensin II, which may be secreted from stretched myocytes, may be partly involved in stretch-induced hypertrophic responses by activating PKC.     

Relationship between 45Ca movements, different calcium components and responses to acetylcholine and potassium in tracheal smooth muscle

Correlations between tension responses elicited with acetylcholine (ACh) and high K+ and corresponding alterations in Ca++ mobilization were obtained in rabbit and canine tracheal smooth muscle. Removal of Ca++ or preincubation with D-600 (50 microM) inhibited responses to K+ (50 or 80 mM) and low ACh (89 nM) and had only a small effect on responses to high ACh (8.9 microM). Conversely, solutions containing Sr++ instead of Ca++ inhibited responses to both concentrations of ACh to a greater degree than were those to K+. Washout of slow component 45Ca into a O-Ca solution was more rapid in rabbit trachea than reported previously for rabbit aorta. Washout of tracheal smooth muscle into an 80.8 mM La -substituted solution at 0.5 degrees C removed superficial (La -accessible) 45Ca and blocked both 45Ca uptake and most 45Ca efflux. D-600, which had no significant effect on control 45Ca uptake in rabbit aortic smooth muscle, decreased 45Ca uptake by 33% in rabbit tracheal smooth muscle. The uptake of 45Ca from the Ca++ binding sites with low affinity for Ca++ was increased by 80 mM K+, 50 mM K+ or 8.9 microM ACh, and the accumulation of Ca++ from the Ca++ binding sites with high affinity for 45Ca was inhibited by Sr++. The stronger effect of either Ca++ removal or D-600 on responses to K+ and the correspondingly greater effect of Sr++ on responses to ACh indicate that different Ca++ stores are present in tracheal smooth muscle. These Ca++ components appear to be qualitatively similar to those present in aortic smooth muscle but they differ quantitatively and are not as readily dissociated as are aortic Ca++ components.     

Phorbol diesters alter the contractile responses of porcine coronary artery

We have studied the effects of activators of the Ca++- and phospholipid-dependent enzyme protein kinase C on isometric tension development by both intact and skinned coronary artery strips. The intact strips contracted upon incubation with 12-O-tetradecanoylphorbol-13-acetate. 12-O-tetradecanoylphorbol-13-acetate produced a leftward shift in the concentration-response relationship for contraction of the tissues by K+, histamine and norepinephrine. Phorbol-12,13-dibutyrate elicited contraction of detergent-skinned artery strips when the free Ca++ concentration in the bathing media was 0.1 microM or greater. This effect was diminished greatly in the presence of polymyxin B, a putative inhibitor of protein kinase C. Phorbol-12,13-dibutyrate shifted the Ca++ concentration-tension response relationship for the skinned tissue to the left. These results are consistent with a role for protein kinase C in regulating the contractile responses of coronary arterial smooth muscle to a variety of stimuli, at least in part by increasing the sensitivity of the contractile apparatus to Ca++.     

Interleukin 1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction.

The cascade of transmembrane signaling events that follow the occupancy of the interleukin 1 receptor remain poorly defined. We examined potential postreceptor transduction systems involved in human recombinant interleukin 1-beta-stimulated prostacyclin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-beta resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A2 activation. Prostacyclin synthesis after interleukin 1-beta (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-beta produced early activation of a phosphoinositide-specific phospholipase C, human umbilical vein endothelium monolayers were labeled with tritiated-2-myoinositol and inositol polyphosphates recovered after interleukin 1-beta stimulation. In contrast to the potent agonist, alpha-thrombin, interleukin 1-beta failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Cai++ secretagogue, IP3, was confirmed in human umbilical vein endothelium monolayers loaded with the Ca++ photoprotein probe aequorin. Basal aequorin luminescence was unaltered after interleukin 1-beta (0 to 2 hours), whereas both alpha-thrombin and Ca++ ionophore A23187 produced rapid rises in Cai++. The intracellular Ca++ antagonist BAPTA and the extracellular Ca++ chelator EGTA produced significant inhibition of interleukin 1-beta-stimulated prostacyclin generation at 4 to 8 hours, suggesting either an indirect inhibitory effect of these agents on phospholipase A2 activity or that an increase in Ca++ may be a late event in the transduction scheme after interleukin 1 stimulation. Interleukin 1-beta-stimulated protein kinase C, phospholipase D, and adenylyl cyclase activities (0 to 4 hours) were unchanged from controls. Despite the absence of increased plasma membrane protein kinase C activity up to 4 hours after interleukin 1, pretreatment of human umbilical vein endothelium monolayers with staurosporine or phorbol myristate acetate (18 hours) to reduce protein kinase C activities, significantly attenuated the interleukin 1-stimulated prostanoid responses at 16 hours but not at 4 hours. Furthermore, short (5 minute) pretreatment with phorbol myristate acetate dramatically augmented interleukin 1-mediated prostacyclin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-beta-mediated endothelial cell phospholipase A2 activity and prostacyclin synthesis occur via a novel transducing pathway that does not involve early activation of phospholipase C, phospholipase D, or adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasma membrane calcium flux, protein kinase C activation and smooth muscle contraction

Isolated perfused rabbit ear arteries contract when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the calcium-activated, phospholipid-dependent protein kinase or C-kinase. Under conditions where the calcium concentration in the perfusate is 1.5 mM and the potassium concentration is 4.8 mM, there is a latent period of 70 +/- 19 min (mean +/- S.E.M., n = 10) between TPA addition and the onset of the contractile response. Once initiated, the contractile response is progressive and sustained. When perfusion conditions are altered in such a way as to modify calcium flux across the plasma membrane (i.e., raising the extracellular calcium concentration to 2.5 mM Ca++, raising the extracellular potassium concentration to 10 mM, and/or preincubating the tissues in media containing 100 nM Bay K 8644, a potent calcium channel agonist), the latency period between TPA addition and initiation of the contractile response is significantly reduced (2.5 mM Ca++, 37 +/- 7 min; 10 mM K+ and 2.5 mM Ca++, 11 +/- 3 min; 100 nM Bay K 8644 and 1.5 mM Ca++, 20 +/- 7 min; 100 nM Bay K 8644 and 2.5 mM Ca2+, 8.5 +/- 1.7 min; 10 mM K+ and 100 nM Bay K 8644, 11 +/- 5 min). Likewise, the combination of 2.5 mM calcium, 100 nM Bay K 8644, and 3.3 microM ouabain results in a contractile response 4.5 +/- 2.0 min after TPA addition (means +/- S.E.M., n = 4). Control tissues (absence of TPA addition) run simultaneously show no contractile responses to the various Ca++ flux regulators even after 90 min of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Specialization in beta-1 and beta-2 adrenoceptor distribution in veins of the rabbit face: relationship to myogenic tone and sympathetic nerve innervation

Studies were performed on several superficial veins from the rabbit face to examine the relationship between beta adrenoceptor subtype distribution, intrinsic myogenic tone and sympathetic nerve innervation. Experiments using selective beta adrenoceptor agonists and antagonists indicate that the dorsal nasal and angularis oculi veins possess a homogeneous population of beta-2 adrenoceptors. Sympathetic nerve stimulation in these segments results only in contraction mediated through postjunctional alpha adrenoceptors. These segments are devoid of intrinsic myogenic tone. In the facial vein, of which both veins are tributaries, both beta-1 and beta-2 adrenoceptors are found. Studies with the beta-1 adrenoceptor antagonist, betaxolol, and beta-2 adrenoceptor antagonist, ICI 118,551, indicate that the prominent relaxation observed in this tissue to sympathetic nerve stimulation is mediated through postjunctional beta-1 adrenoceptors. At physiological temperatures, the facial vein possesses a marked intrinsic myogenic tone that is inhibited by this beta-1 adrenoceptor-mediated sympathetic activity. Considering the anatomical relationship between these vessels and the unique association between beta adrenoceptor subtype, intrinsic myogenic tone and sympathetic innervation, it is possible that facial blood redistribution in the rabbit can be markedly affected by sympathetic nerve stimulation. Such a process could have an important role in cranial thermoregulation.     

Inhibitory effects of aminoglycosides on renal protein phosphorylation by protein kinase C

Aminoglycosides such as neomycin are commonly prescribed antibiotics; however, there is associated serious damage to the kidney. We examined the effect of aminoglycoside antibiotics on renal protein phosphorylation and found that neomycin selectively inhibited Ca++-activated, phospholipid-dependent phosphorylation of 88-kDa protein in cell lysates of the rabbit kidney. Fifty percent inhibition of phosphorylation of this protein occurred with 5 X 10(-5) to 1 X 10(-4) M neomycin. In living PtK2 cells, neomycin dose-dependently inhibited 12-O-tetradecanoyl-phorbol-13-acetate-induced phosphorylation of 88 K Da protein. This drug also inhibited phosphorylation of exogenous protein catalyzed by protein kinase C, isolated from rabbit kidney in vitro. In contrast, neomycin had little or no inhibitory effect on cyclic GMP-dependent protein kinase, cyclic AMP-dependent protein kinase, casein kinase I, casein kinase II and Ca++-calmodulin-dependent myosin light chain kinase. Whereas activity of protein kinase C was inhibited 65% by neomycin (0.1 mM) at pH 5 to 7, inhibition decreases to 33% at pH 8 and to zero at pH 9. The potencies of a series of aminoglycoside antibiotics to inhibit the kinase agreed well with number of ionizable amino groups of compounds (gamma = 0.99) and this also approximates their known nephrotoxic potential; amikacin less than or equal to kanamycin less than gentamycin less than or equal to tobramycin less than neomycin. As aminoglycoside antibiotics present in the kidney after administration of toxicological doses (10(-2) M) will inhibit the effects of protein kinase C, the aminoglycoside antibiotics-induced nephrotoxicity is discussed in relation to inhibition of intracellular protein kinase C.     

3H]BAY K 8644, a 1,4-dihydropyridine Ca++ channel activator: characteristics of binding to high and low affinity sites in cardiac membranes

The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.     

Cyclical mechanical stretch enhances angiopoietin-2 and Tie2 receptor expression in cultured human umbilical vein endothelial cells

Endothelial cells are essential for neovascularization. Angiopoietins and Tie receptors are required for a normal vasculature. How cyclical mechanical stretch affects the expression of components of the angiopoietin system is not known. In this study, we investigated the regulation of angiopoietins and Tie receptors by cyclical mechanical stretch in cultured human umbilical vein endothelial cells (HUVECs). HUVECs grown on a flexible membrane base were stretched by vacuum to 20% elongation, at 60 cycles/min. The levels of angiopoietin-2 protein began to increase as early as 2 h after stretch was initially applied, reached a maximum of 2.7-fold over the control value by 6 h. The Tie2 receptor protein showed the same pattern as Ang-2. These increases in angiopoietin-2 and Tie2 receptor proteins at 6 h were blocked by the addition (30 min before stretch) of the protein kinase C inhibitor G?6976 (16 nM) or the tyrosine kinase inhibitor herbimycin A (24 microM). Similar to protein expression, the levels of angiopoietin-2 and Tie2 receptor mRNAs in HUVECs increased 3.1-fold and 2.5-fold respectively after stretch for 6 h. These increases were also blocked by G?6976 or herbimycin A. Cyclical mechanical stretch increased (and G?6976 or herbimycin A abrogated these increases) the immunohistochemical labelling of angiopoietin-2 and Tie2 receptor after a 6 h stretch. The levels of angiopoietin-1 and Tie1 receptor proteins, mRNAs and immunohistochemical staining were unaffected by cyclical mechanical stretch. Thus cyclical mechanical stretch activates the expression of angiopoietin-2 and the Tie2 receptor, but not angiopoietin-1 or the Tie1 receptor, in cultured HUVECs. This mechanical effect is probably mediated by the tyrosine kinase and protein kinase C pathways.     

Angiotensin II amplifies arterial contractile response to norepinephrine without increasing Ca++ influx: role of protein kinase C.

We investigated whether the enhanced contractile response to norepinephrine caused by a subthreshold concentration of angiotensin II was associated with an increased 45Ca++ influx or net uptake. Rabbit facial artery segments were mounted isometrically to measure the 45Ca++ influx and net uptake in response to norepinephrine. The contractile response to norepinephrine (3 microM) in the presence of angiotensin II (0.1 nM) was 149.5 +/- 7.4% of control. This response amplification was not associated with changes in norepinephrine-induced 45Ca++ influx or net uptake. Angiotensin II also potentiated the contractile response to caffeine obtained in a Ca(++)-free buffer containing ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (2 mM) to 148.0 +/- 4.8% of control. In both cases, the amplification was prevented by pretreatment with either staurosporine (10 nM) or calphostin C (100 nM), two inhibitors of protein kinase C. We conclude that angiotensin II potentiation of norepinephrine-induced vascular tone occurs in the absence of changes in stimulated Ca++ entry. This potentiation may be due to an increase in intracellular sensitivity to Ca++, possibly mediated by protein kinase C.     

Mechanism of vascular smooth muscle contraction by sodium fluoride in the isolated aorta of rat and rabbit.

The purpose of this study was to determine the cellular basis for fluoride ion (F-)-induced contractions of isolated aortic rings from both the rat and the rabbit. The F- contractions were not affected by endothelial denudation but were enhanced in the presence of A (0.1 or 1.0 mM) added to the bathing Krebs' solution. The contractile effect of F- also was not modified by bathing with Ca(++)-free + ethylene glycol bis(b-aminoethylether)-N,N-tetracetic acid Krebs' solution or nifedipine (10 microM), but was attenuated by inorganic (Cd++, Co++ and Ni++) Ca++ antagonists in normal and Ca(++)-free Krebs' media. Bis(o-aminophenoxy)-ethane-N-N-N'-N'-tetraacetic acid, ryanodine and intracellular Ca++ modulators, respectively, caused 36.1 +/- 6.1%, 16.4 +/- 6.8% and 52.3 +/- 7.3% inhibition of the contractile response to F- in a Ca(++)-free media while causing near complete inhibition of norepinephrine-induced contractions. F- contractions were also inhibited by the calmodulin antagonists W-7 and calmidazolium (IC50 = 23.0 +/- 7.0 and 45.0 +/- 10.0 microM, respectively). On the other hand, the protein kinase C antagonists staurosporine and H-7 potently (IC50 = 0.016 +/- 0.007 and 1.1 +/- 0.5 microM, respectively) inhibited the fluoride-induced contractions. Aortic rings from the rabbit were similarly potently antagonized by the protein kinase C inhibitors, however, K(+)-induced contractions were also equally sensitive to these agents in both rat and rabbit tissues. The putative phospholipase C inhibitor neomycin was significantly less effective (IC50 = 13.0 +/- 5.0, 0.44 +/- 0.09 and 0.89 +/- 0.40 mM) at inhibiting F- than norepinephrine and KCl contractile effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of endothelium-dependent vasodilation in pressurized cerebral and small mesenteric resistance arteries of the rat.

We compared endothelial responses to calcium-mobilizing agents in mesenteric and cerebral resistance arteries of the rat. Middle cerebral and small mesenteric arteries were mounted in a pressure myograph, and myogenic responses were recorded. The effects of acetylcholine (ACh), bradykinin, substance P, histamine, A23187, cyclopiazonic acid (CPA), and sodium nitroprusside were investigated in both arteries with myogenic tone in the absence and presence of nitric oxide synthase and cyclooxygenase inhibitors. The effects of raised potassium, K(+) channel blockers, and arachidonic metabolism inhibition were examined on the nitric oxide (NO) synthase/cyclooxygenase inhibitor-resistant dilation induced by ACh and CPA. Cerebral arteries display a high level of myogenic reactivity compared with mesenteric arteries. In cerebral arteries, only bradykinin and substance P induced endothelium-dependent dilation. The observed dilation was solely related to the activation of the NO pathway. However, in mesenteric arteries, all of the vasoactive agents induced endothelium-dependent dilation. A combination of NO, cyclooxygenase-derived prostanoids, and a factor with endothelium-derived hyperpolarizing factor-like properties was responsible for the observed vasodilation. NO and cyclooxygenase derivatives were able to compensate for each other in the CPA-induced endothelium-dependent vasodilation when one of the two pathways was blocked. Moreover, small Ca(2+)-activated K(+) channels and a combination of both large and small Ca(2+)-activated K(+) channels were implicated in the endothelium-derived hyperpolarizing factor-like component of dilation to ACh and CPA, respectively. Finally, the results suggest that the pathway by which agonists raise intracellular calcium concentration may determine the nature of the endothelial secretory product.     

Interactions between several rare earth ions and calcium ion in vascular smooth muscle.

The effects of lutetium (Lu+++), europium (Eu+++) and neodymium (Nd+++) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Tension responses to norepinephrine were markedly inhibited by all three rare earth ions; K+-induced responses and those to histamine were partially blocked. Addition of 1.5 mM Lu+++, Eu+++ or Nd+++ to the bathing solution reduced 45Ca uptake (Nd+++ greater than Eu+++ greater than greater than Lu+++), produced a maintained increase in 45Ca efflux from strips of media-intimal layer washed out in a 0-Ca++ solution (Nd+++ greater than Eu+++ greater than Lu+++), and, when present during 45Ca incubation (before 45Ca washout), altered the release of 45Ca elicited by addition of 1.5 mM Ca++ during the 45Ca washout (inhibition by Nd+++ greater than Eu+++ greater than Lu+++). The 3-fold greater uptake of 45Ca in a 0-Ca++ and 1.5 mM Lu+++ solution than in an equivalent Sr++ solution results from a decreased effect of Lu+++ upon the slower 45Ca washout component. The actions of Lu+++ were quantitatively similar to those of low La+++ concentrations (0.05-0.10 mM), whereas those of Nd+++ and Eu+++ resembled effects obtained with equimolar concentrations of La+++. Thus, the three rare earth ions produce qualitatively similar alterations in 45Ca uptake and superficial binding. The difference observed can be attributed to variations in their affinity for superficial Ca++ binding sites and in their ability to block Ca++ uptake.     

Stretch-induced VEGF expression in the heart.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in vascular development and angiogenesis. Recently we have observed increased VEGF expression in the normal myocardium after myocardial infarction in a rat heart. This study was designed to explore the mechanism responsible for this increase in VEGF expression. Induction of myocardial stretch in an isolated perfused Langendorff preparation by inflation of an intraventricular balloon to an end-diastolic load of 35 mmHg for 30 min resulted in a nearly sixfold increase in VEGF message level not only in the chamber subjected to stretch (left ventricle) but also in the unstretched right ventricle, thus raising the possibility of a soluble factor mediating stretch- induced induction of VEGF expression. This was further confirmed by demonstrating that coronary venous effluent collected from the stretched heart and used to perfuse isolated hearts in which no balloon was present was able to induce VEGF expression in these normal hearts. Inhibition of TGF-beta activity using a neutralizing antibody, but not antagonists/inhibitors of endothelin and angiotensin II, eliminated stretch-induced increase in VEGF expression. Staurosporine, a protein kinase C inhibitor, also blocked stretch-induced increase of VEGF expression. Measurement of TGF-beta concentration in the perfusate demonstrated increased amounts of the cytokine after myocardial stretch, and addition of TGF-beta protein to the perfusion buffer resulted in increased VEGF expression in control hearts. These results suggest that stretch-induced increase of VEGF expression in the heart is mediated at least in part by TGF-beta.     

Phorbol ester-induced stress and myosin light chain phosphorylation in swine carotid medial smooth muscle

The mechanisms by which activators of protein kinase C (PKC) stimulate contractile responses in arterial smooth muscle is not known. In this study, we assessed the relative contribution of CA(++)-dependent and independent pathways in mediating phorbol ester-induced 20 kdalton myosin light chain (MLC)-phosphorylation and force in medial smooth muscle strips from swine carotid artery. Phorbol 12,13-dibutyrate (PDB; 10(-7)M)-stimulated stress development was associated with a significant increase in the fraction of phosphorylated MLC, from 0.08 +/- 0.02 to 0.24 +/- 0.02 after 30 min of stimulation. Under conditions of Ca++ depletion, which normally do not support Ca++/calmodulin-dependent activation of myosin light chain kinase (MLCK) by physiological stimuli, PDB-induced contractile responses were reduced significantly. However, after Ca2++ depletion, PDB (10(-6) M; 30 min) still caused an increase in MLC-phosphorylation from 0.10 +/- 0.02 at rest to 0.19 +/- 0.03. Preincubation with nifedipine (10(-7) M) had no significant effect on contractile responses to PDB, indicating that Ca++ influx through nifedipine-sensitive voltage channels did not contribute significantly to the observed Ca++ dependency of the PDB responses. Staurosporine (0.1-0.3 microM), a putative PKC inhibitor, significantly inhibited PDB-induced contractile and MLC phosphorylation responses. Tonic histamine (3 microM)- and KCl-induced contractile and MLC-phosphorylation responses were inhibited by the same concentrations of staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific actions of gallium on norepinephrine-induced tension and associated 45Ca movements in rabbit aortic smooth muscle

Gallium ion (Ga) dose-dependently (60-360 microM) inhibited contractions induced by norepinephrine (NE, 1 microM) in rabbit aortic (and media intimal) strips, but did not affect contractions elicited with high K+ (80 mM) solution. The initial phasic portion of the NE-induced response was either unaffected or only slightly (less than 10%) reduced, but the tonic portion of the response was inhibited completely by higher concentrations (greater than or equal to 300 microM) of Ga . In resting muscles, the equilibrated (90 min) 45Ca uptake was not altered by Ga (360 microM). Also, 45Ca efflux from either high- or low-affinity Ca++ binding sites was unaltered by Ga . The effects of Ga (360 microM) on 45Ca retained after a subsequent 60-min washout at 0.5 degrees C in an isosmotic (80.8 mM) La solution were also examined. High affinity La -resistant 45Ca released by NE (1 microM) was not altered by Ga . Under conditions favoring low affinity Ca++ uptake, 45Ca retention in control and K+-treated muscles was not changed by Ga , but the additional incremental 45Ca uptake associated with NE (in the presence of high K+) was blocked. Thus, Ga appears to have a selective inhibitory action on NE-associated 45Ca uptake without affecting either resting and high K+-induced 45Ca uptake or that 45Ca fraction released by NE. This action may result from a selective blockade by Ga of receptor-linked Ca++ channels in rabbit aortic smooth muscle.     

Evidence for pH as an important parameter to control when studying the contractility of isolated rabbit cerebral arteries

Summary— Isolated rabbit basilar (BA), middle (MCA) and posterior (PCA) cerebral arteries mounted in an isometric Mulvany myograph and stretched to a given level of resting tension, developed a spontaneous slow-rising contraction. This tone presented the main features of a classical myogenic tone since it was not suppressed by repetitive washings, was not dependent on the presence of endothelium and was markedly influenced by the concentration of extracellular calcium. In addition, we observed that the occurrence of myogenic tone was dependent on the pH of the medium buffer. Decreasing pH of the Krebs solution from 7.54 to 6.81 by lowering NaHCO, concentration from 25 to 5 mM. reduced the amplitude of the myogenic tone developed by the arteries. We investigated the influence of such changes of pH on the contractile response to potassium chloride (KCl) and to 5-hydroxytryptamine (5-HT). We demonstrated that the contractile response to KCl (124 mM) was not affected by the pH of the organ bath whereas the maximum contraction to 5-HT (10 μM) was significantly affected in BA but not in MCA and PCA. Furthermore, we found that three consecutive concentration-response curves to 5-HT were reproducible when obtained at pH 7.15 and 7.30 for BA and MCA. In PCA, 5-HT-induced responses were reproducible at pH 7.30 whereas the sensitivity of the repeated response curves to 5-HT was reduced at pH 7.15. We also noted a larger variability of the response to 5-HT for the three arteries at this pH. Thus, it appeared that bathing rabbit cerebral arteries in a buffer medium at pH 7.30 is suitable for studying their reactivity to 5-HT. These results highlight the importance of controlling the pH of medium buffer when studying in vitro the reactivity of rabbit cerebral arteries. In addition, they define appropriate conditions to minimize the myogenic tone and to allow vigorous and reproducible contractile responses to 5-hydroxytryptamine.