The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta

We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 +/- 5.1% and 22.3 +/- 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.     

Mental retardation, epilepsy, short stature, and skeletal dysplasia: confirmation of the Gurrieri syndrome

AIM: To study the relationship between the calcium and the release of platelet-activating factor (PAF) from rat peritoneal macrophages (PM?) stimulated by lipopolysaccharides (LPS). METHODS: The effects of tetrandrine (Tet), Fura 2-AM, and Bay k 8644 on the PAF release from rat PM? was investigated by the bio-assay of PAF. RESULTS: LPS stimulated PM? to release PAF, without increasing intracellular Ca2+ of PM?, Tet at 0.1, 1.0, 10, 100 mumol.L-1 and Fura 2-AM at 0.01, 0.1, 1.0, 10 mumol.L-1 could dose-dependently decrease the release of PAF (9.8 +/- 1.2, 6.5 +/- 1.6, 4.7 +/- 0.8, 3.4 +/- 0.4 microgram.L-1 and 9.2 +/- 1.7, 5.2 +/- 1.3, 3.7 +/- 0.4, 3.2 +/- 0.3 microgram.L-1, respectively, no drugs 11.8 +/- 1.2 micrograms.L-1), Bay k 8644 at 1.0, 5.0, 10 mumol.L-1 could dose-dependently increase the release of PAF (13.2 +/- 1.7, 16.2 +/- 1.4, 17.6 +/- 1.5 micrograms.L-1), and the effects of Tet and Fura 2-AM were completely or partly reversed by Bay k 8644 at 5.0 mumol.L-1. CONCLUSION: Although LPS did not increase intracellular Ca2+ of PM?, intracellular Ca2+ was necessary for PAF release from rat PM? stimulated by LPS.     

Overexpression of the cardiac Na+/Ca2+ exchanger increases susceptibility to ischemia/reperfusion injury in male, but not female, transgenic mice

Influx of Ca2+ into myocytes via Na+/Ca2+ exchange may be stimulated by the high levels of intracellular Na+ and the changes in membrane potential known to occur during ischemia/reperfusion. This increased influx could, in turn, lead to Ca2+ overload and injury. Overexpression of the cardiac Na+/Ca2+ exchanger therefore may increase susceptibility to ischemia/reperfusion injury. To test this hypothesis, the hearts of male and female transgenic mice, overexpressing the Na+/Ca2+ exchange protein, and hearts of their wild-type littermates, were perfused with Krebs-Henseleit buffer and subjected to 20 minutes of ischemia and 40 minutes of reperfusion. Preischemic left ventricular developed pressures and +dP/dtmax, as well as -dP/dtmin, were higher in the male transgenic hearts compared with wild-type, implying a role for Na+/Ca2+ exchange in the contraction, as well as the relaxation, phases of the cardiac beat. Postischemic function was lower in male transgenic than in male wild-type hearts (7+/-2% versus 32+/-6% of preischemic function), but there was no difference between female transgenic and female wild-type hearts, both at approximately 30% of preischemic function. To assess whether this male/female difference was due to female-specific hormones such as estrogen, the hearts of bilaterally ovariectomized and sham-operated transgenic females were subjected to the same protocol. The functional recoveries of ovariectomized female transgenic hearts were lower (17+/-3% of preischemic function) than those of wild-type and sham-operated transgenic females. The lower postischemic functional recovery in the male transgenic and female ovariectomized transgenic hearts correlated with lower recoveries of the energy metabolites, ATP and phosphocreatine, as measured by 31P nuclear magnetic resonance spectroscopy. Alternans were observed during reperfusion in male transgenic and female ovariectomized transgenic hearts only, consistent with intracellular Ca2+ overload. Western analyses showed that alterations in the expression of the Na+/Ca2+ exchange or L-type Ca2+ channel proteins were not responsible for the protection observed in the female transgenic hearts. In conclusion, in males, overexpression of the Na+/Ca2+ exchanger reduced postischemic recovery of both contractile function and energy metabolites, indicating that the Na+/Ca2+ exchanger may play a role in ischemia/reperfusion injury. From the studies of females, however, it appears that this exacerbation of ischemia/reperfusion injury by overexpression of the Na+/Ca2+ exchanger can be overcome partially by female-specific hormones such as estrogen.     

Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs

Coronary resistance arteries isolated from exercise-trained pigs have been shown to exhibit enhanced myogenic reactivity (J. M. Muller, P. R. Myers, and M. Harold Laughlin. J. Appl. Physiol. 75: 2677-2682, 1993). The purpose of this study was to test the hypothesis that exercise training results in enhanced vasoconstrictor responses of these arteries to all vasoconstrictor stimuli [specifically acetylcholine (ACh), endothelin-1 (ET-1), KCl, and the Ca2+ channel-agonist Bay K 8644]. Female Yucatan miniature swine were trained (Trn) on a motor-driven treadmill (n = 16) or remained sedentary (Sed, n = 15) for 16-20 wk. Arteries 50-120 micron in diameter were isolated and cannulated with micropipettes, and intraluminal pressure was set at 60 cmH2O throughout experiments. Vasoreactivity was evaluated by examining constrictor responses to increasing concentrations of ACh (10(-9) to 10(-4) M), ET-1 (10(-10) to 10(-8) M), KCl (bath replacement with isotonic physiological saline solution containing 30 or 80 mM), and Bay K 8644 (10(-9) to 10(-6) M). Constricted diameters are expressed relative to the passive diameter observed after 100 microM SNP. All four constrictors produced similar decreases in diameter in arteries from both groups [ACh: 0.52 +/- 0.07 (Trn) and 0.54 +/- 0,06 (Sed); ET-1: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); KCl: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); Bay K 8644: 0.86 +/- 0.05 (Trn) and 0. 76 +/- 0.05 (Sed)]. Present results combined with previous observations indicate that exercise training does not alter vasoconstrictor responses of porcine coronary resistance arteries but specifically increases myogenic reactivity. Thus the underlying cellular mechanisms for myogenic tone are altered by training but not receptor-mediated mechanisms (ACh and ET-1) nor voltage-gated Ca2+ channels (KCl and Bay K 8644) in coronary resistance arteries.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9 mM KCl or veratridine (10 microM) were inhibited by spantide (10 microM), a tachykinin receptor antagonist, and capsaicin (10 microM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. omega-Conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K 8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9 mM KCl, and enhanced contractions induced by 15.9 mM KCl and veratridine (2 microM). omega-Agatoxin IVA (0.3 microM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K 8644. These results suggest that, in the rabbit iris sphincter muscle. L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.     

Regulation of prolactin, thyrotropin subunit, and gonadotropin subunit gene expression by pulsatile or continuous calcium signals

We investigated the importance of calcium (Ca2+) influx in increasing the steady state concentrations of mRNAs coding for the pituitary peptides PRL, alpha, and TSH, LH, and FSH beta-subunits. Adult female rat pituitaries were dissociated, plated for 48 h, then inserted into perifusion chambers. Secretory responses were measured after 2 and 22 h of perifusion, and after 24 h, the cells were recovered, total RNA was extracted, and mRNAs were assayed by dot blot hybridization. The first experiment examined the effect of the Ca2+ channel blocker verapamil (100 microM) on the stimulatory action of pulsatile TRH (4 nM; 60-min interval) or GnRH (100 pM; 60-min interval) on pituitary mRNAs. TRH pulses induced a significant increase (49-56%) in PRL, alpha, and TSH beta mRNAs. Similarly, GnRH pulses stimulated a rise in alpha (64%) and FSH beta (50%) mRNAs, but not LH beta. The effects of pulsatile TRH or GnRH were eliminated when verapamil was added to the medium, suggesting that Ca2+ influx is critical to the stimulatory action of TRH or GnRH. The second experiment examined the effect of pulsatile vs. continuous increases in intracellular Ca2+ on pituitary mRNA expression. Pulsatile Ca2+ signals were produced by giving 60-min pulses of 50 mM KCl, Bay K 8644 (10 microM), or Bay K 8644 (10 microM; in the presence of 10 mM KCl in the injectate) and vehicle pulses to controls. Continuous increases in intracellular Ca2+ were induced by perifusion with medium containing the Ca2+ ionophore A23187 (20 microM), and these groups were compared to that receiving continuous verapamil. Pulsatile increases in Ca2+ influx (KCl or Bay K 8644) stimulated significant elevations in all mRNAs studied (36-74% increase vs. controls), with the exception of TSH beta. The magnitude of the mRNA responses to pulsatile Ca2+ (vs. controls) was similar to that observed after TRH and GnRH pulses. In contrast, only LH beta was increased by A23187 (42% increase vs. controls; P < 0.05). PRL and alpha mRNAs were selectively diminished by A23187 (57% and 83% decreases vs. controls, respectively; P < 0.05) and verapamil (67% and 60%; P < 0.05). The data show that expression of these pituitary genes is regulated by Ca2+ and that a pulsatile Ca2+ signal is required to stimulate PRL, alpha, and FSH beta (but not LH beta).(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro and in vivo vascular responses to the L-type calcium channel activator, Bay K 8644, in rats with cirrhosis

A substance which increases the entry of extracellular calcium into arterial smooth muscle may decrease cirrhosis-induced vasodilation. The aim of the present study was to measure the effects of the L-type Ca2+ channel activator, Bay K 8644, on the haemodynamics of rats with cirrhosis. Vascular reactivity to this substance was also investigated. Splanchnic and systemic haemodynamic responses to Bay K 8644 (50 microg/kg) were measured in cirrhotic and normal rats. Contraction induced by 0.1 micromol/L Bay K 8644 was measured in arterial rings (aorta and superior mesenteric artery) from cirrhotic and normal rats. In cirrhotic rats, Bay K 8644 significantly decreased portal pressure (15%) and portal tributary blood flow (24%), significantly increased portal territory vascular resistance (54%) and did not significantly change hepatocollateral vascular resistance. Bay K 8644 significantly increased arterial pressure (7%) and systemic vascular resistance (24%) and did not change the cardiac index. In normal rats, Bay K 8644 significantly increased vascular resistance (150%) in portal, hepatocollateral and systemic territories and significantly decreased the cardiac index (44%). Changes in portal territory, hepatocollateral and systemic vascular resistances were significantly less marked in cirrhotic than in normal rats. In rings from the aorta and superior mesenteric artery, Bay K 8644-induced contraction was significantly lower in cirrhotic than in normal rats. In conclusion, in rats with cirrhosis, Bay K 8644 administration reduced vasodilation in splanchnic and systemic arteries and did not affect hepatocollateral vascular resistance. The Bay K 8644-induced reduction in splanchnic vasodilation caused a decrease in portal hypertension. This study also shows that Bay K 8644-induced vascular contraction was less marked in cirrhotic than in normal rats, in systemic and splanchnic vascular beds.     

Canine bronchial sustained contraction in Ca2+-free medium: role of intracellular Ca2+

We evaluated whether cartilage was a source of Ca2+ and the possible role of Ca2+ recycling in the sustained bronchial contraction (SBC) induced by carbachol (Cch) in Ca2+-free medium. Canine first-order bronchi were studied with cartilage and epithelium (+CAR + EPI) and without these structures individually (-CAR + EPI and +CAR - EPI) or together (-CAR - EPI). After cartilage removal (-CAR - EPI or -CAR + EPI) Cch produced a transient contraction in Ca2+-free medium. Removal of the epithelium alone had minor effects on the magnitude of the SBC but increased the effect of removal of cartilage to diminish the SBC. Bronchial strips with cartilage were able to respond to Cch with lower Ca2+ concentrations (10-100 microM) than could dissected preparations. Preincubation with BAY K 8644 (30-1000 nM) or 60 mM KCl or -CAR - EPI tissues converted the transient contractions to Cch in Ca2+-free medium to sustained contractions. In microelectrode studies, 50 nM Cch induced membrane oscillations in solutions with 2.5 mM Ca2+ in bronchial preparations, plus or minus cartilage, and in undissected tissues in Ca2+-free medium but not in -CAR - EPI tissues. Preincubation with 1 microM BAY K 8644 in Ca(2+)-free medium restored these oscillations in -CAR - EPI tissues. The release of 45Ca2+ from cartilage was too rapid to provide a reservoir of Ca2+ to support multiple SBCs in Ca2+-free medium. Moreover, in the Ca2+-free medium (with 10 nM Ca2+ after tissue +CAR + EPI incubation) excitatory junction potentials rapidly disappeared. Addition of 1 microM nifedipine or 1 mM EGTA during the SBC of +CAR + EPI tissues produced complete relaxation. A transient contraction to Cch occurred with prior addition of nifedipine. Inhibition of the sarcoplasmic reticulum Ca2+ pump by tissue incubation with cyclopiazonic acid (CPA; 10 microM), or briefly with 1 mM EGTA significantly diminished the SBC induced by Cch in Ca2+-free medium. CPA and EGTA together abolished the Cch-induced SBC. Thus, cartilage plays a more complex role than as a Ca2+ reservoir to support the SBC induced by Cch in Ca2+-free solution; its removal affects the process supporting SBCs involving intracellular Ca2+ storage and Ca2+ entrance through voltage-dependent channels.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.     

Bay K 8644 increases resting Ca2+ spark frequency in ferret ventricular myocytes independent of Ca influx: contrast with caffeine and ryanodine effects

Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.     

Nitric oxide, a possible mediator of 1,4-dihydropyridine-induced photorelaxation of vascular smooth muscle

1. In rat aortic tissues pre-contracted with phenylephrine, certain 1,4-dihydropyridines (DHPs) such as Bay K 8644 (0.1 microM), PN 202791 (1 microM), RK 30 (1 microM), NI 104 (1 microM) and NI 105 (1 microM) enhanced photoactivated relaxations (photorelaxation or PR) whereas NI 72, NI 85, NI 99, NI 102, amlodipine, felodipine, nifedipine and nimodipine were inactive. 2. The PR inducing effects of Bay K 8644 were mimicked by the diabetogenic agent, streptozotocin (STZ). 3. Solutions of Bay K 8644 which had been irradiated for various periods of time initiated light independent transient relaxations followed by contractile responses in aortic tissue partially contracted with phenylephrine. With exposure times to light of 30 to 120 min, the intensity of the relaxation response to irradiated Bay K 8644 increased from 26 +/- 3.3 to 71 +/- 3.7% of the maximum contractile response to phenylephrine (n = 5). Conversely the contractile responses decreased, from 84.2 +/- 4.1 to 19.8 +/- 10.4% of the maximum contractile response to phenylephrine (n = 5). 4. Superoxide ions, generated by incubation of xanthine (2mM) plus xanthine oxidase (10 mu ml-1) in physiological saline solution (PSS) NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 12.5 and glucose 11.1 (mM) for 1 h. reduced the PR induced by DHPs, STZ, and also NO-induced relaxations of rat aortic preparations. 5. Direct measurements of NO indicate that, following exposure to a polychromatic light source, equimolar concentrations (0.1 mM) of the DHP compounds that enhance PR, as well as STZ, photodegrade to release NO (25 +/- 2-40.3 +/- 5.9 nmol min-1, n = 6). 6. Structure-activity studies indicate that a nitro group at the -3 position of the dihydropyridine ring is essential for DHPs to support PR. 7. These data suggest that the photodegradation of DHPs and STZ leading to the release of NO provides the primary cellular process underlying the PR response.     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

Rehabilitation of Guillain-Barré syndrome

In this study, we evaluated the effects of vitamin E on the vascular reactivity and structure of thoracic aorta from streptozotocin (STZ)-diabetic rats. Plasma glucose, cholesterol, and triglyceride concentrations in rats were increased markedly by STZ-diabetes. The thiobarbituric acid (TBA) reactivity level as an index of lipid peroxidation was higher in both plasma and aorta of STZ-diabetic rats compared with controls. The rings of thoracic aorta with or without endothelium were mounted in organ chambers for measurement of isometric tension and were contracted by a single dose (10-5 mol/L) and then cumulative doses of noradrenaline ([NA] 10(-9) to 10(-5) mol/L). Pretreatment with methylene blue (MB) or removal of the endothelium resulted in a similar degree of enhancement in NA-induced contraction of control rings. STZ-diabetes increased the fast and slow components of NA-induced contraction in all experiments. The maximal contractile response of aorta to NA was also augmented by STZ-diabetes, whereas the sensitivity (pD2) remained unaltered. STZ-diabetes resulted in significant increases in the maximum contractile response and sensitivity of aorta to KCl. STZ-diabetic rats showed a significant reduction in the percentage of endothelial response (PER). A group of diabetic rats was treated from the time of diabetes induction with a 0.5% dietary supplement of vitamin E. Vitamin E supplementation of STZ-diabetic rats eliminated accumulation of lipid peroxides and returned plasma triglycerides toward normal levels. Diabetes-induced abnormal contractility and endothelial dysfunction were significantly but not completely prevented by vitamin E treatment. The endothelium-independent relaxation response to sodium nitroprusside (SNP) was not affected by diabetes or vitamin E treatment. Electron microscopic examination of thoracic aorta revealed that normal tissue organization was disrupted in STZ-diabetic rats, and that vitamin E treatment can protect the morphological integrity of aorta against STZ-diabetes. The results suggest the following: (1) The increased triglycerides/lipid peroxides may be an important reason for morphological or functional disruption of endothelium and enhanced activation of contractile mechanisms of vascular smooth muscle in STZ-diabetic rats. Both contribute to an increased responsiveness of diabetic aorta to vasoconstrictor agents. (2) Vitamin E treatment of STZ-diabetic rats can prevent the development of abnormal contractility and structure and endothelial dysfunction in aorta. (3) The triglyceride- and/or lipid peroxidation-lowering effect of vitamin E may be crucial for the protective effect of this vitamin on the vasculature.     

Understanding the interactions between physician assistants and their supervising physicians in the provision of patient care

Both contractile and relaxant responses to tetrapentylammonium ions (TPA+) were studied in rat isolated mesenteric artery. TPA+ (5-10 micromol/l) caused a sustained increase of muscle tension. The contractile effect of TPA+ (10 micromol/l) was dependent upon the presence of extracellular Ca2+ but independent of the presence of endothelium. TPA+ (10-50 micromol/l) induced biphasic contraction, and the amplitude of peak and sustained tension decreased with increasing TPA+ concentration. TPA+ (100-300 micromol/l) only produced monophasic contraction. TPA+ (50 micromol/l) abolished the transient contraction induced by caffeine (10 mmol/l) or phenylephrine (1 micromol/l) in the absence of extracellular Ca2+. Nifedipine and verapamil concentration-dependently reduced the TPA+-induced contraction with respective IC50 values of 1.34 +/- 0. 24 and 9.46 +/- 1.36 nmol/l, these values were similar to 1.35 +/- 0. 21 and 16.07 +/- 1.71 nmol/l, respectively, for the inhibitory effects of nifedipine and verapamil on the high K+ (60 mmol/l)-induced contraction. TPA+ (>10 micromol/l) concentration-dependently reduced the phenylephrine (1 micromol/l)-, U46619 (30 nmol/l)-, endothelin I (10 nmol/l)- and high K+ (60 mmol/l)-induced sustained tension with respective IC50 values of 53. 7 +/- 9.5, 31.9 +/- 5.3, 30.9 +/- 3.4 and 20.9 +/- 2.8 micromol/l. The present results indicate that TPA+ at low concentrations could contract the arterial smooth muscle probably through promoting Ca2+ influx. At higher concentrations (>20 micromol/l), TPA+ relaxes arterial smooth muscle probably through inhibition of both nifedipine-sensitive Ca+ channels and internal Ca2+ release. TPA+, unlike other quaternary ammonium ions, could therefore act at multiple sites in arterial smooth muscle.     

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.     

Relationship between nifedipine sensitivity of aortae and blood pressure of stroke-prone spontaneously hypertensive rats

1. We have previously described an increased sensitivity to inhibition by nifedipine of noradrenaline-induced contractures of blood vessels in hypertension. In this study we have investigated whether changes in blood pressure (BP) change the sensitivity to nifedipine and K+ of aortic rings from normotensive (Wistar-Kyoto rats, WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). 2. SHRSP were treated with: hydralazine plus hydrochlorothiazide; captopril plus hydrochlorothiazide; hydralazine plus guanethidine; or captopril alone. WKY rats were treated with deoxycorticosterone acetate (DOCA) and NaCl. Treatment commenced from 5 weeks of age and continued until 13-15 weeks. 3. The SHRSP treatments produced similar reductions in BP, and the BP of all the treated groups were significantly lower than the mean BP of untreated SHRSP (201.0 +/- 7.7 mmHg). The mean BP of the treated WKY rats (134.2 +/- 7.6 mmHg) was significantly higher than the mean BP of the untreated WKY rats (86.8 +/- 7.4 mmHg). 4. An area-under-curve (AUC) analysis of the inhibitory effects of nifedipine on responses of aortae to noradrenaline showed no differences between treated and untreated SHRSP groups (overall mean 40.6 +/- 1.9% and 43.4 +/- 3.4% inhibition of control AUC, respectively), or between DOCA-salt treated WKY and untreated WKY groups (58.8 +/- 5.9 and 64.8 +/- 2.3, respectively). Noradrenaline-induced contractures of aortae from all SHRSP groups were significantly more sensitive to inhibition by nifedipine than aortae from both WKY groups. 5. The molar concentration of agonist required to evoke 50% of the maximum response (EC50) values for potassium chloride (KCl) were significantly increased in the aortae of all treated SHRSP groups in comparison to those from untreated SHRSP (treated SHRSP groups, 15.53 +/- 0.68 mmol/L vs untreated SHRSP group, 11.36 +/- 1.10 mmol/L). The EC50 values for KCl for the aortae from the DOCA-treated WKY rats were significantly less than those from aortae of the untreated WKY (11.80 +/- 0.80 and 17.08 +/- 1.50 mmol/L, respectively). 6. We conclude that reduction (in SHRSP) or increase (in WKY) of the BP has no effect on the sensitivity of aortic smooth muscle to the inhibitory effects of nifedipine on responses to noradrenaline, suggesting that alterations in voltage-dependent Ca2+ mechanisms may be a primary phenomenon in the SHRSP. In contrast, the fact that sensitivity to KCl changes in the treated SHRSP and WKY aortae suggests such sensitivity is secondary to the BP and thus a separate phenomenon from voltage-dependent Ca2+ mechanisms.     

Pharmacological studies on alterations in contractile reactivity in aortas isolated from experimental diabetic rats

The present study was undertaken to determine how the responses to contractile agents are altered in aortas from rats with streptozotocin-induced diabetes and to explore the possible mechanisms of the altered responses in diabetes. Rats were given an intravenous injection of 45 mg/kg streptozotocin. Eight to 12 weeks after treatment, aortas were isolated and set up for measurement of isometric tension. Diabetic aortas exhibited significantly lesser contractions in response to high K+ than those from age-matched controls. Furthermore, the Ca2+ channel agonist Bay K 8644 was not able to consistently contract diabetic aortas even when they were partially depolarized by an elevation of the extracellular K+ concentration to 15 mM where the agonist produced concentration-dependent contractions in all control aortas. On the other hand, the contractile responses to norepinephrine, 5-hydroxytryptamine, endothelin-1 and U46619 were significantly enhanced in diabetic rat aortas. All of the enhanced responses of diabetic aortas were completely eliminated in the presence of the Ca2+ channel antagonist nifedipine. The contractile responses of aortas from both control and diabetic rats to these agonists were abolished or strongly inhibited by the protein kinase C inhibitor staurosporine, and no significant difference was found in the magnitude of the contractile responses of aortas between control and diabetic rats to the agonists in the presence of staurosporine. In diabetic aortas, the protein kinase C activators phorbol 12, 13-dibutyrate and 12-O-tetradecanoylphorbol 13-acetate elicited a delayed, sharply developing rise in tension following the initial, gradually developing contraction, while these agents produced only the initial, slowly developing contraction in control aortas. As a result, the contractions induced by phorbol esters were greater in diabetic aortas than in controls. The enhanced contractile responses of diabetic aortas to phorbol esters were not observed in Ca(2+)-free medium or in the presence of nifedipine. In Ca(2+)-free medium, the transient contraction induced by caffeine was significantly diminished in diabetic aortas, in contrast to the phasic contraction by norepinephrine which was similarly observed in control and diabetic aortas. These results indicate that the extracellular Ca(2+)-dependent contractions elicited by receptor activation are enhanced in aortas from diabetic rats, and this is presumably related to a greater influx of Ca2+ through transmembrane Ca2+ channels as a consequence of increased protein kinase C-activated processes. On the other hand, the contractions associated with depolarization-evoked activation of Ca2+ channels are diminished in diabetic aortas, possibly due to an alteration in activation of the channels by membrane depolarization, and Ca(2+)-induced Ca2+ release from intracellular stores appears to be impaired in diabetes.     

Effect of age on in vitro magnesium modulation of vascular reactivity in the rat aorta

Vascular reactivity and the effect of various magnesium (Mg) concentrations on it, were studied in aortic rings from adult (4-month-old) and aged (24-month-old) male Sprague-Dawley rats. Contraction induced by CaCl2 of the aortae incubated in high potassium PSS containing 1.2 mM Mg was greater in aged than in adult rats. Low Mg (0.1 mM) decreased CaCl2-induced contraction in the aortae from adult rats more than in those from aged rats. High Mg (4.8 mM) attenuated CaCl2-induced contraction in the aged but not in the adult rats. Acetylcholine- and isoproterenol-induced relaxation of the aortae incubated in normal PSS (1.2 mM) was less pronounced in aged than in adult rats, whereas sodium nitroprusside-induced relaxation was similar in both groups. Low Mg did not modify acetylcholine- and sodium nitroprusside-induced relaxation in adult and aged rats. With high Mg, acetylcholine- and sodium nitroprusside-induced relaxation was increased in both groups. The increasing effect of high Mg on acetylcholine-induced relaxation was however greater in aorta from aged rats. Low Mg decreased isoproterenol-induced relaxation in the adult but not in the aged group, whereas high Mg increased it in both groups of rats. When endothelium was intact, Mg-induced relaxation was less in aged than in adult rats. When endothelium was disrupted, relaxation was similar in both groups . Mg Removal produced an endothelium-dependent relaxation, which was significantly lower in the aged rats. In conclusion, the functional alterations of vascular smooth muscle and endothelium observed with aging modify the modulatory role of Mg on aortic responsiveness.     

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca2+ in rat aorta

We investigated the effects of palmitoyl-L-alpha-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K+, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective alpha2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10(-6) M to 10(-4) M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K+ - and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10(-5) M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10(-5) M) markedly augmented the increase in both cytosolic free Ca2+ ([Ca2+]i) and contractile tension induced by UK14,304, high K+ and PMA. Nicardipine (10(-7) M) and 10(-6) M Ro-31-8220 (?1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-++ +indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca2+]i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K+ -induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca2+]i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.     

GTP-binding protein regulates the contractile response elicited by the phorbol ester (PMA)-induced activation of protein kinase C in the isolated rat aorta

1. The aim of the present study was to investigate the involvement of GTP-binding protein in the contractile response induced by activation of protein kinase C (PKC) in isolated rat aorta. The rats were treated with islet-activating protein (IAP) for 4 days prior to the experiments. 2. In the aorta from control rats, phorbol 12-myristate 13-acetate (PMA) produced biphasic contractions; twitch contraction superimposed on the slowly developing contraction. The twitch contraction was abolished by the removal of external Ca2+ or by treatment with nicardipine. In the aorta pretreated with IAP, PMA produced only a slowly developing contraction, and no twitch contraction was induced. 3. The application of Ca2+ to aortic strips in a Ca(2+)-free solution, that had been treated with 10(-6) M PMA caused concentration-dependent contraction, and the contraction was completely inhibited by IAP. 4. Pretreatment with IAP inhibited Ca(2+)-induced contraction of the aorta in Ca(2+)-free medium in the presence of 10(-6) M clonidine, but did not affect the Ca(2+)-induced contraction in the medium treated with 10(-6) M phenylephrine and 10(-7) M nicardipine. 5. These results suggest that the activation of PKC by PMA produces biphasic contractions in the rat aorta. The twitch contraction may be induced by the activation of voltage-dependent Ca(2+)-channels and the activation may be regulated by IAP-sensitive GTP-binding protein.