[Effect of hypochloric acid on Escherichia coli biofilm and the clinical efficacy of hypochloric acid for wounds with Escherichia coli infection].

Objective: To investigate the effect of hypochloric acid on Escherichia coli biofilm and the clinical efficacy of hypochloric acid for wounds with Escherichia coli infection. Methods: One strain of Escherichia coli with the strongest bacterial biofilm forming ability among the strains isolated from specimens in 25 patients (16 males and 9 females, aged 32-67 years) from five clinical departments of the 940th Hospital of the Joint Logistic Support Force was collected for the experimental study from September to December 2019. The Escherichia coli was cultured with hypochloric acid at 162.96, 81.48, 40.74, 20.37, 10.18, 5.09, 2.55, 1.27, 0.64, and 0.32 µg/mL respectively to screen the minimum bactericidal concentration (MBC) of hypochloric acid. The Escherichia coli was cultured with hypochloric acid at the screened MBC for 2, 5, 10, 20, 30, and 60 min respectively to screen the shortest bactericidal time of hypochloric acid. The biofilm formation of Escherichia coli was observed by scanning electron microscopy at 6, 12, 24, 48, 72, and 96 h of incubation, respectively. After 72 h of culture, hypochloric acid at 1, 2, 4, 8, and 16 times of MBC was respectively added to Escherichia coli to screen the minimum biofilm eradicate concentration (MBEC) of hypochloric acid against Escherichia coli. After hypochloric acid at 1, 2, 4, and 8 times of MBEC and sterile saline were respectively added to Escherichia coli for 10 min, the live/dead bacterial staining kit was used to detect the number of live and dead cells, with the rate of dead bacteria calculated (the number of samples was 5). From January to December 2020, 41 patients with infectious wounds meeting the inclusion criteria and admitted to the Department of Burns and Plastic Surgery of the 940th Hospital of Joint Logistic Support Force of PLA were included into the prospective randomized controlled trial. The patients were divided into hypochloric acid group with 21 patients (13 males and 8 females, aged (46±14) years) and povidone iodine group with 20 patients (14 males and 6 females, aged (45±19) years) according to the random number table. Patients in the 2 groups were respectively dressed with sterile gauze soaked with hypochloric acid of 100 µg/mL and povidone iodine solution of 50 mg/mL with the dressings changed daily. Before the first dressing change and on the 10th day of dressing change, tissue was taken from the wound and margin of the wound for culturing bacteria by agar culture method and quantifying the number of bacteria. The amount of wound exudate and granulation tissue growth were observed visually and scored before the first dressing change and on the 3rd, 7th, and 10th days of dressing change. Data were statistically analyzed with one-way analysis of variance, Dunnett-t test, independent sample t test, Mann-Whitney U test, Wilcoxon signed-rank test, chi-square test, or Fisher's exact probability test. Results: The MBC of hypochloric acid against Escherichia coli was 10.18 µg/mL, and the shortest bactericidal time of hypochloric acid with MBC against Escherichia coli was 2 min. Escherichia coli was in a completely free state after 6 and 12 h of culture and gradually aggregated and adhered with the extension of culture time, forming a mature biofilm at 72 h of culture. The MBEC of hypochloric acid against Escherichia coli was 20.36 µg/mL. The Escherichia coli mortality rates after incubation with hypochloric acid at 1, 2, 4, and 8 times of MBEC for 10 min were significantly higher than that after incubation with sterile saline (with t values of 6.11, 25.04, 28.90, and 40.74, respectively, P<0.01). The amount of bacteria in the wound tissue of patients in hypochloric acid group on the 10th day of dressing change was 2.61 (2.20, 3.30)×104 colony forming unit (CFU)/g, significantly less than 4.77 (2.18, 12.48)×104 CFU/g in povidone iodine group (Z=2.06, P<0.05). The amounts of bacteria in the wound tissue of patients in hypochloric acid group and povidone iodine group on the 10th day of dressing change were significantly less than 2.97 (2.90, 3.04)×106 and 2.97 (1.90, 7.95)×106 CFU/g before the first dressing change (with Z values of 4.02 and 3.92, respectively, P<0.01). The score of wound exudate amount of patients in hypochloric acid group on the 10th day of dressing change was significantly lower than that in povidone iodine group (Z=2.07, P<0.05). Compared with those before the first dressing change, the scores of wound exudate amount of patients in hypochloric acid group on the 7th and 10th days of dressing change were significantly decreased (with Z values of -3.99 and -4.12, respectively, P<0.01), and the scores of wound exudate amount of patients in povidone iodine group on the 7th and 10th days of dressing change were significantly decreased (with Z values of -3.54 and -3.93, respectively, P<0.01). The score of wound granulation tissue growth of patients in hypochloric acid group on the 10th day of dressing change was significantly higher than that in povidone iodine group (Z=2.02, P<0.05). Compared with those before the first dressing change, the scores of wound granulation tissue growth of patients in hypochloric acid group on the 7th and 10th days of dressing change were significantly increased (with Z values of -3.13 and -3.67, respectively, P<0.01), and the scores of wound granulation tissue growth of patients in povidone iodine group on the 7th and 10th days of dressing change were significantly increased (with Z values of -3.12 and -3.50, respectively, P<0.01). Conclusions: Hypochloric acid can kill Escherichia coli both in free and biofilm status. Hypochloric acid at a low concentration shows a rapid bactericidal effect on mature Escherichia coli biofilm, and the higher the concentration of hypochloric acid, the better the bactericidal effect. The hypochloric acid of 100 µg/mL is effective in reducing the bacterial load on wounds with Escherichia coli infection in patients, as evidenced by a reduction in wound exudate and indirect promotion of granulation tissue growth, which is more effective than povidone iodine, the traditional topical antimicrobial agent.

Diagnosis of solitary pulmonary lesions with intravoxel incoherent motion diffusion-weighted MRI and semi-quantitative dynamic contrast-enhanced MRI.

AIM: To evaluate the diagnostic value of intravoxel incoherent motion (IVIM) diffusion-weighted (DWI) magnetic resonance imaging (MRI) and semi-quantitative dynamic contrast-enhanced MRI (DCE-MRI) to help diagnose indeterminate solitary pulmonary lesions (SPLs) and the subgroups of lung cancer (LC), and to explore the relationship between IVIM and DCE-MRI. MATERIALS AND METHODS: Sixty-four consecutive patients (44 male, 20 female; age, 52.77±10.46 years) from February 2014 to September 2016 with SPLs, were involved in this prospective study. Total apparent diffusion coefficient (ADCtotal), tissue diffusivity (D), pseudo-diffusion coefficient (D*), perfusion fraction (F), maximum enhancement ratio (MER), Tmax, slope, and washout were compared between the lung cancer (LC) and benign group and among the subtypes of LC. Time-intensity curves (TICs) were drawn. Receiver operating characteristic (ROC) curves were constructed to estimate the diagnostic performance. The correlation of both tools was assessed. RESULTS: ADCtotal, D, and Tmax were significantly higher for benignity than for LC (p=0.005, p=0.002 and p<0.001 respectively). D* and slope were significantly higher in LC than benignity (p=0.005 and p=0.011, respectively). D and Tmax had the highest sensitivity and accuracy, respectively. A combination of D and Tmax improved the sensitivity to 90.5%, the specificity to 86.4%, and the accuracy to 89.1%. Poor correlations were found between parameters derived from IVIM and DCE-MRI. ADCtotal values of SCC and SCLC were found to be significantly lower compared with that in adenocarcinoma. CONCLUSION: Both IVIM-DWI and DCE-MRI were useful for discriminating benignity from LC. ADCtotal was helpful for distinguishing adenocarcinoma and non-adenocarcinoma. A combination of DCE-MRI and IVIM could provide a robust method to determine the microstructural characteristics of SPLs.

Acupuncture for patients with chronic functional constipation: A randomized controlled trial.

BACKGROUND: Acupuncture is used to treat chronic functional constipation (CFC) in China, despite limited evidence. We aim to assess the effectiveness and safety of acupuncture in managing CFC. METHODS: A multicenter randomized controlled trial was performed involving 684 patients with CFC; the patients were randomly allocated to receive He acupuncture (n = 172), Shu-mu acupuncture (n = 171), He-shu-mu acupuncture (n = 171), or oral administration of mosapride (n = 170). Sixteen sessions of acupuncture were given in the treatment duration of 4 weeks. The primary outcome was the change in spontaneous bowel movements (SBMs) at week 4 (at the end of treatment) compared to baseline. The secondary outcomes included stool consistency (Bristol scale), the degree of straining during defecation, and adverse events. KEY RESULTS: The SBMs increased in all the four groups at week 4, and the magnitude of increase was equivalent in the four groups (He acupuncture, 2.7 [95% CI, 2.3-3.1]; Shu-mu acupuncture, 2.7 [95% CI, 2.3-3.0]; He-shu-mu acupuncture, 2.2 [95% CI, 1.9-2.5]; and mosapride, 2.4 [95% CI, 2.0-2.9]; P = .226). However, the change in SBMs at week 8 was significantly smaller in mosapride group (1.4 [95% CI, 1.0-1.8]) than the three acupuncture groups (2.4 [95% CI, 2.1-2.7], 2.3 [95% CI, 1.9-2.7], 2.1 [95% CI, 1.7-2.5] in He, Shu-mu, and He-shu-mu group, respectively, P = .005). CONCLUSIONS & INTERFERENCES: The three acupuncture treatments were as effective as mosapride in improving stool frequency and stool consistency in CFC, but the magnitude of the treatment effect is unknown due to the lack of sham acupuncture control.

Effect of feed selenium supplementation on milk selenium distribution and mozzarella quality.

In the present study, the effect of feed Se supplementation on the Se content of raw milk and mozzarella cheese as well as the effect on cheese quality and functionality were determined. The Se milk was produced by supplying dairy cow feed with Se yeast (0.3mg of Se/kg of dry matter), resulting in a Se concentration in milk of 35.81µg/L. The fat, casein, and whey protein of Se milk were separated by ultracentrifugation, and the Se content was determined by atomic absorption spectroscopy. The Se distribution in different milk fractions of fat, casein, and whey protein were 9.82, 45.56, and 44.62%, respectively. The Se mozzarella cheese was made by Se milk, and the composition and texture of Se cheese did not significantly differ from that of the control. However, the functional properties (meltability, flowability, and stretchability) of the Se cheese were better after 8 wk of storage. Moreover, the pH and water activity were lower in Se cheese, which decreased the total plate count. The Se content in mozzarella cheese was 4 fold higher than that in milk, and Se was found in the whey, hot water, and brine collected during cheesemaking. Organic and inorganic Se was found in the Se cheese after 8 wk of storage, and most Se peptides detected after storage were Se-Met and Se-Cys. The results of this study show that feed Se supplementation can improve the Se content of milk and cheese without affecting mozzarella cheese quality.

Enhanced susceptibility-weighted angiography (ESWAN) of cerebral arteries and veins at 1.5 Tesla.

OBJECTIVE: Enhanced susceptibility-weighted angiography (ESWAN) is a three-dimensional (3D) multi-echo gradient-echo sequence which consists of both magnitude and phase images. This study aims to demonstrate the feasibility of ESWAN for the depiction of both cerebral arteries and veins at 1.5 T by comparing with time-of-flight (TOF) MR angiography (MRA) and MR venography (MRV). METHODS: 13 healthy volunteers underwent both ESWAN and 3D-TOF-MRA examinations. Among them, nine volunteers underwent an additional two-dimensional-TOF-MRV examination. With regard to the ESWAN sequence, both maximum intensity projection (MIP) and minimum intensity projection (mIP) images were reconstructed and compared with MIP reconstructions of the TOF MRA and the TOF MRV. RESULTS: Concerning the depiction of the constituent segments of the Circle of Willis, as well as A1, A2, A3 (segments of the anterior cerebral artery), M1, M2 (segments of the middle cerebral artery), P1 and P2 (segments of the posterior cerebral artery), the value of the ESWAN MIP was comparable to that of the TOF MRA without regard to visualization of branches, vessel homogeneity and wall irregularities or slight stenosis. ESWAN-mIP visualized more deep cerebral veins than TOF MRV in this study. CONCLUSION: By use of either mIP reconstruction of a long echo data set or MIP reconstruction of a short echo data set, ESWAN allows simultaneous visualization of both cerebral veins and proximal segments of intracerebral arteries at 1.5 T. ADVANCES IN KNOWLEDGE: ESWAN acquires multiple images at different echo times corresponding to different T2* weightings, wherein a short echo TOF-MRA data set and a long echo susceptibility-weighted imaging-MRV data set are obtained simultaneously.

Assessment of lateral geniculate nucleus atrophy with 3T MR imaging and correlation with clinical stage of glaucoma.

BACKGROUND AND PURPOSE: Although previous animal studies have shown structural changes in ocular hypertension such as atrophy of the LGN, such changes have not been thoroughly studied in human glaucoma patients nor correlation made with clinical stage. Our aim was to investigate prospectively LGN atrophy in patients with POAG using 3T MR imaging and correlation with the clinical stage of disease. MATERIALS AND METHODS: Twenty-six patients with known POAG and 26 age-matched healthy volunteers were included in this institutional review board-approved study. All subjects underwent imaging on a 3T MR imaging system with a PD and GM sequence. LGN height and volume were measured by 2 blinded neuroradiologists. Measurements were compared and correlated with clinical glaucoma severity as assessed by static threshold visual field parameters. RESULTS: Average maximum LGN height in patients with glaucoma on PD images was 4.36 ± 0.61 mm (right) and 4.31 ± 0.61 mm (left), significantly less (P < 10⁻³) than respective measurements of 5.05 ± 0.41 and 4.99 ± 0.41 mm in volunteers. With the GM sequences, such respective measurements were also less (P < 10⁻³) in patients with glaucoma (4.20 ± 0.71 mm right, 4.00 ± 0.85 mm left) versus respective measurements in volunteers (4.88 ± 0.51 mm right, 4.77 ± 0.47 mm left). Average LGN volumes in the patient group were 98.0 ± 27.2 mm³ (right) and 93.7 ± 25.8 mm³ (left) with the PD sequence versus respective measurements of 85.2 ± 27.1 and 80.5 ± 23.6 mm³ with the GM sequence. All height and volume measurements were greater in volunteers (P < 10⁻³). In the patient group, both maximum height and volume of the LGN with both sequences were significantly correlated with cumulative clinical glaucoma stage (P < .05). CONCLUSIONS: MR imaging measurements of LGN height and volume are diminished in patients with glaucoma, with the extent of atrophy correlating to clinical stage, suggesting a novel imaging marker of disease severity.

Human embryonic stem cell-derived cardiomyocytes engraft but do not alter cardiac remodeling after chronic infarction in rats.

Previous studies indicated that, in an acute myocardial infarction model, human embryonic stem cell-derived cardiomyocytes (hESC-CM) injected with a pro-survival cocktail (PSC) can preserve contractile function. Because patients with established heart failure may also benefit from cell transplantation, we evaluated the physiological effects of hESC-CM transplanted into a chronic model of myocardial infarction. Intramyocardial injection of hESC-CM with PSC was performed in nude rats at 1 month following ischemia-reperfusion. The left ventricular function of hESC-CM injected rats was evaluated at 1, 2 and 3 months after the cell injection procedure and was compared to 3 control groups (rats injected with serum-free media, PSC only, or non-cardiac human cells in PSC). Histology at 3 months revealed that human cardiomyocytes survive, develop increased sarcomere organization and are still proliferating. Despite successful engraftment, both echocardiography and MRI analyses showed no significant difference in left ventricular structure or function between these 4 groups at any time point of the study, suggesting that human cardiomyocytes do not affect cardiac remodeling in a rat model of chronic myocardial infarction. When injected into a chronic infarct model, hESC-CM can engraft, survive and form grafts with striated cardiomyocytes at least as well as was previously observed in an acute myocardial infarction model. However, although hESC-CM transplantation can attenuate the progression of heart failure in an acute model, the same hESC-CM injection protocol is insufficient to restore heart function or to alter adverse remodeling of a chronic myocardial infarction model.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

beta 2-adrenergic receptor-induced p38 MAPK activation is mediated by protein kinase A rather than by Gi or gbeta gamma in adult mouse cardiomyocytes.

Increasing evidence shows that stimulation of beta-adrenergic receptor (AR) activates mitogen-activated protein kinases (MAPKs), in addition to the classical G(s)-adenylyl cyclase-cAMP-dependent protein kinase (PKA) signaling cascade. In the present study, we demonstrate a novel beta(2)-AR-mediated cross-talk between PKA and p38 MAPK in adult mouse cardiac myocytes expressing beta(2)-AR, with a null background of beta(1)beta(2)-AR double knockout. beta(2)-AR stimulation by isoproterenol increased p38 MAPK activity in a time- and dose-dependent manner. Inhibiting G(i) with pertussis toxin or scavenging Gbetagamma with betaARK-ct overexpression could not prevent beta(2)-AR-induced p38 MAPK activation. In contrast, a specific peptide inhibitor of PKA, PKI (5 microm), completely abolished the stimulatory effect of beta(2)-AR, suggesting that beta(2)-AR-induced p38 MAPK activation is mediated via a PKA-dependent mechanism, rather than by G(i) or Gbetagamma. This conclusion was further supported by the ability of forskolin (10 microm), an adenylyl cyclase activator, to elevate p38 MAPK activity in a PKI-sensitive manner. Furthermore, inhibition of p38 MAPK with SB203580 (10 microm) markedly enhanced the beta(2)-AR-mediated contractile response, without altering base-line contractility. These results provide the first evidence that cardiac beta(2)-AR activates p38 MAPK via a PKA-dependent signaling pathway, rather than by G(i) or Gbetagamma, and reveal a novel role of p38 MAPK in regulating cardiac contractility.

Spontaneous activation of beta(2)- but not beta(1)-adrenoceptors expressed in cardiac myocytes from beta(1)beta(2) double knockout mice.

Although ligand-free, constitutive beta(2)-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac beta(2)-AR, it is unclear whether the dominant cardiac beta-AR subtype, beta(1)-AR, shares the ability of spontaneous activation. In the present study, we expressed human beta(1)- or beta(2)-AR via recombinant adenoviral infection in ventricular myocytes isolated from beta(1)beta(2)-AR double knockout mice, creating pure beta(1)-AR and beta(2)-AR systems with variable receptor densities. A contractile response to a nonselective beta-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral beta(1)-AR or adenoviral beta(2)-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10-1000) led to a dose-dependent expression of beta(1)- or beta(2)-AR with a maximal density of 1207 +/- 173 (36-fold over the wild-type control value) and 821+/-38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of beta(1)-AR and beta(2)-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, beta(2)-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal beta(2)-AR density. These effects were specifically reversed by a beta(2)-AR inverse agonist, ICI 118,551 (10(-7) M). In contrast, overexpression of beta(1)-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged beta(1)-AR inverse agonist, CGP 20712A (10(-6) M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute beta(2)-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is beta-AR subtype specific because beta(1)-AR does not exhibit agonist-independent spontaneous activation.

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

Characterization of subtype of alpha 1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed.

AIM: To characterize the subtype of alpha 1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed. METHODS: The potencies (pA2 values determined by Schild plot) of alpha 1-adrenoceptor-selective antagonists were determined by isolated vasoconstrictive experiment. The pKi values were determined by 125I-BE 2254 binding from the cloned alpha 1A-, alpha 1B-, and alpha 1D-adrenoceptor, stably expressed in human embryonic kidney (HEK) 293 cells. RESULTS: The pA2 values for alpha 1A-adrenoceptor-selective antagonists, RS-17053, WB 4101, 5-methyl-urapidil, and the alpha 1D-adrenoceptor-selective antagonist, BMY 7378, were 8.98 +/- 0.28, 9.16 +/- 0.20, 8.69 +/- 0.02, and 6.03 +/- 0.26, respectively, with the slope not different from unity. The pA2 values of the above antagonists correlated well with the binding pKi values only for alpha 1A-adrenoceptors (r = 0.97), but not for alpha 1B-adrenoceptors (r = 0.52) and alpha 1D-adrenoceptors (r = 0.04). The concentration-vasopressor response curve for norepinephrine was not affected by pretreatment with chloroethylclonidine (Chl) 50 mumol.L-1 for 30 min. CONCLUSION: Only alpha 1A-adrenoceptors mediate the norepinephrine-induced vasopressor response in perfused rat mesenteric vascular bed.

Tyrosine kinase participates in alpha 1A-adrenoceptor-mediated increase of intracellular calcium in human embryo kidney 293 cells.

AIM: To determine the role of protein-tyrosine kinase (PTK) in alpha 1A-adrenoceptor-mediated increase of [Ca2+]i (intracellular calcium) in human embryo kidney (HEK) 293 cells expressed alpha 1A-adrenoceptor. METHODS: Effects of two PTK inhibitors: genistein and tyrphostin, were investigated on the increase of [Ca2+]i by using Fura-2, The activity of PTK was measured and the accumulation of [3H] InsPs were observed. RESULTS: Norepinephrine stimulated a rapid increase in [Ca2+]i to (371 +/- 31) nmol.L-1 in HEK 293 cells. Norepinephrine-induced increase of [Ca2+]i was inhibited by the tyrosine kinase inhibitors quercetin and tyrphostin by 23.8% and 21.4%, respectively, but the accumulation of [3H]InsPs induced by norepinephrine was not. The activity of the plasma-associated tyrosine kinase was increased to (1.73 +/- 0.72)-fold over the control by norepinephrine 10 mumol.L-1. The norepinephrine-activated PTK was inhibited by calphostin C and depletion of intra- and extra-cellular Ca2+. CONCLUSION: The PTK participates in mobilization of Ca2+ mediated by alpha 1A-adrenoceptors in HEK 293 cell lines.

Ca2+-dependence of vasoconstriction mediated by alpha1A-adrenoceptors in perfused rat hindlimb: a pharmacological approach.

We investigated the source of Ca2+ for the vasoconstriction mediated by alpha1a-adrenoceptors in perfused rat hindlimb in functional studies. The noradrenaline (NA)-induced maximum response was decreased by 92% following perfusion with Ca2+-free medium. Depletion of intracellular Ca2+-stores with repeatedly application of caffeine and NA in Ca2+-free medium resulted in complete abolishment of NA-response. Nifedipine concentration-dependently inhibited NA-contraction with a maximum inhibition of 65%. The residual nifedipine-insensitive response was further inhibited by Cd2+. Following depletion of Ca2+ stores with cyclopiazonic acid in Ca2+ free medium for 30 min, the NA-response obtained by re-admission of Ca2+ was decreased by 80%. However, re-introduction of Ca2+ to NA-treated tissues in Ca2+-free medium without prior treatment with cyclopiazonic acid normalizes the NA-response. These results suggest that the NA-contraction in this preparation is mediated largely via an influx of extracellular Ca2+, of which the majority utilizes L-type calcium channels. Only a small portion of the contractile response to NA is derived from intracellular stores, which probably also play a modulatory role on Ca2+ influx.

Tyrosine kinases participate in alpha 1A-adrenoceptor-mediated vasoconstriction in perfused rat hindlimb.

AIM: To determine whether or not tyrosine kinase is involved in the signal transduction of alpha 1A-adrenoceptors. METHODS: Effects of various pharmacological probes on norepinephrine (NE)-induced vasopressor responses were determined in the perfused rat hindlimb. RESULTS: The putative tyrosine kinase inhibitors, genistein, and tyrphostin, significantly inhibited the vasopressor responses induced by NE but not that induced by KCl. The protein-tyrosine-phosphatase inhibitor, sodium orthovanadate, selectively potentiated the vasopressor responses induced by NE. Neither genistein nor tyrphostin had effect on the contraction elicited by phorbol 12-myristate 13-acetate. In contrast, both genistein and tyrphostin attenuated the vasopressor responses evoked by NaF. CONCLUSION: The genistein- and tyrphostin-sensitive tyrosine kinases participate in alpha 1A-adrenoceptor-mediated vasoconstriction in perfused rat hindlimb.

[Effects of naphthylmethyl isoquinoline on contraction and calcium flux of rabbit vascular strips in vitro].

Naphthylmethyl isoquinoline (NI) 1-30 mumol.L-1 inhibited the contraction of rabbit vascular smooth muscle in vitro induced by KCl, CaCl2, and norepinephrine (NE). NI 0.3-30 mumol.L-1 blocked 45Ca2+ influx process in vascular smooth muscle of aorta, mesenteric and femoral arteries by addition of KCl and NE. NI 3-10 mumol.L-1 had no effect on 45Ca2+ efflux from aorta at resting state. These results suggest that the relaxing effect of NI on rabbit blood vessels may be relevant to the inhibition of Ca2+ influx into vascular smooth muscle.