Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 microm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.     

Action and mechanism of action of veratrine on renin secretion from rat kidney slices

It is well known that norepinephrine released from the renal nerves stimulates the secretion of renin by a beta adrenergic mechanism. In the present experiments, we investigated the effects of renin secretion of veratrine, which depolarizes nerve terminals and thereby causes transmitter release. The rat renal cortical slice preparation was used. Veratrine (10-200 microM) stimulated renin secretion in a concentration-dependent manner. Veratrine-stimulated secretion was antagonized by timolol (0.9 and 9.0 microM) and by tetrodotoxin (0.5 and 5.0 microM), a sodium channel blocker. Neither drug abolished completely the stimulatory effect of veratrine. Moreover, veratrine stimulated renin secretion in slices prepared from previously denervated kidneys; this response was not antagonized by timolol. These results are consistent with the hypothesis that veratrine stimulates renin secretion by at least two mechanisms. One component probably consists of veratrine-induced depolarization of renal nerve terminals, release of norepinephrine and activation of juxtaglomerular cell beta adrenergic receptors; the other component appears to be independent of nerve terminals in the preparation. We conclude that the tetrodotoxin-sensitive component of veratrine-stimulated renin secretion in this preparation is an in vitro model of renal nerve-stimulated renin secretion; it should be useful in investigating substances which affect renin secretion by presynaptic modulation of transmitter release.     

Cellular mechanism of stimulation of renin secretion by the mercurial diuretic mersalyl.

The aim of the present study was to elucidate the cellular mechanism by which the mercurial diuretic mersalyl stimulates renin secretion in rabbit renal cortical slices in vitro. The stimulatory effect of mersalyl on renin secretion was rapid, reversible and concentration dependent. The stimulation was not dependent on the presence of ions such as Na+, Cl- and Ca++, and it was unaffected by inhibitors of Na+/K+/2Cl- cotransport, such as bumetanide and furosemide. However, the stimulation was blocked and reversed by thiols, such as L-cysteine and dithiothreitol. Furthermore, the maximal stimulatory effect of mersalyl on renin secretion was not additive to that produced by the non-diuretic mercurial sulfhydryl reagent P-chloromercuriphenylsulfonate nor to that produced by the non-mercurial diuretic sulfhydryl reagent, ethacrynic acid. These results support the hypothesis that mersalyl stimulates renin secretion by forming a reversible mercaptide bond with sulfhydryl groups, located perhaps on the plasma membrane of juxtaglomerular cells. These particular sulfhydryl groups appear to have no functional role in the diuretic action of mersalyl.     

Connexin 43-Enhanced Suicide Gene Therapy Using Herpesviral Vectors

Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.     

Diuretic therapies in low renin and normal renin essential hypertension.

This study was designed to compare the effectiveness of spironolactone, hydrochlorothiazide, and combined spironolactone-hydrochlorothiazide therapy in patients with low renin and those with normal renin essential hypertension. Patients with low renin hypertension had a greater hypotensive response to each regimen (p less than 0.001). Low renin patients responded equally to both spironolactone and to hydrochlorothiazide, and in low renin but not in normal renin patients reduction of blood pressure correlated with weight loss. These results suggest that a volume factor, not specifically related to increased mineralocorticoid production, contributes to the pathogenesis of low renin essential hypertension.     

Uterine blood flow and uterine renin secretion

Experiments were carried out in pregnant nephrectomized rabbits to determine the relationship between uterine blood flow and uterine renin secretion. Uterine blood flow was measured by the percentage distribution of radioactive microspheres injected into the left ventricle which lodged in uterus and placenta, and cardiac output was measured by dye dilution. In 40 animals, 24 hr after nephrectomy, uterine blood flow was 4.7+/-0.4% of cardiac output and absolute flow 32.4+/-3 ml/100 g per min. Plasma renin activity (PRA) in uterine vein, 994+/-182 ng/100 ml per hr, was higher than in carotid artery, 832+/-143 (P < 0.025). With reduction of uterine blood flow from 4.7+/-0.5 to 1.95+/-0.3% of cardiac output and absolute flow from 30.8+/-4.6 to 8.8+/-2 ml/100 g per min, uterine vein PRA rose from 1434+/-234 to 4430+/-300 (P < 0.001), and carotid artery PRA from 1009+/-200 to 2300+/-350 (P < 0.01). Hemorrhagic hypotension caused uterine vein PRA to increase from 913+/-293 to 3638+/-1276 (P < 0.001) and carotid artery PRA from 774+/-252 to 1730+/-433 (P < 0.01). Uterine blood flow expressed as a percentage of cardiac output remained constant after hemorrhage, 5.5+/-0.9 and 6.3+/-0.8%, although absolute flow fell from 37+/-7.7 to 29+/-3.6 ml/100 g per min because of the large fall in cardiac output which occurred.Angiotensin, 10 ng/kg per min, caused no significant change in blood pressure or cardiac output but increased uterine blood flow from 4.1+/-0.6 to 8.4+/-1% (P < 0.005) of cardiac output with absolute flow increasing from 37.4+/-7 to 73.2+/-10 ml/100 g per min (P < 0.001). The increase in uterine blood flow during angiotensin was abolished by the prior administration of propranolol. Isoproterenol, 0.5 mu/min, increased uterine blood flow from 3.5+/-0.6 to 6.4+/-1.2% of cardiac output (P < 0.02) with absolute flow increasing from 25+/-5 to 51+/-12 ml/100 g per min (P < 0.05). Norepinephrine, 500 ng/min, caused no significant change in uterine blood flow.These findings suggest that uterine renin might be involved in regulating uterine blood flow, secretion being increased in response to a reduction in flow with the resultant rise in circulating or local angiotensin, through beta adrenergic stimulation, increasing uterine blood flow.     

Breast cancer chemotherapy induces vascular dysfunction and hypertension through a NOX4-dependent mechanism

Cardiovascular disease is the major cause of morbidity and mortality in breast cancer survivors. Chemotherapy contributes to this risk. We aimed to define the mechanisms of long-term vascular dysfunction caused by neoadjuvant chemotherapy (NACT) and identify novel therapeutic targets. We studied arteries from postmenopausal women who had undergone breast cancer treatment using docetaxel, doxorubicin, and cyclophosphamide (NACT) and from women with no history of such treatment matched for key clinical parameters. We explored mechanisms in WT and Nox4^–/– mice and in human microvascular endothelial cells. Endothelium-dependent, NO-mediated vasodilatation was severely impaired in patients after NACT, while endothelium-independent responses remained normal. This was mimicked by a 24-hour exposure of arteries to NACT agents ex vivo. When applied individually, only docetaxel impaired endothelial function in human vessels. Mechanistic studies showed that NACT increased inhibitory eNOS phosphorylation of threonine 495 in a Rho-associated protein kinase–dependent (ROCK-dependent) manner and augmented vascular superoxide and hydrogen peroxide production and NADPH oxidase activity. Docetaxel increased expression of the NADPH oxidase NOX4 in endothelial and smooth muscle cells and NOX2 in the endothelium. A NOX4 increase in human arteries may be mediated epigenetically by diminished DNA methylation of the NOX4 promoter. Docetaxel induced endothelial dysfunction and hypertension in mice, and these were prevented in Nox4^–/– mice and by pharmacological inhibition of Nox4 or Rock. Commonly used chemotherapeutic agents and, in particular, docetaxel alter vascular function by promoting the inhibitory phosphorylation of eNOS and enhancing ROS production by NADPH oxidases.     

间隙连接蛋白43、c-Fos在足月妊娠子宫平滑肌中的表达及意义

【目的】从转录和翻译水平研究间隙连接蛋白43（Connexin 43,Cx43）基因、c-Fos基因及其蛋白在足月妊娠子宫平滑肌中的表达,以及它们之间的相关性,探讨其在分娩发动中的作用。【方法】应用核酸原位杂交技术和SP免疫组化法,检测10例足月未临产、10例足月临产和10例非孕正常子宫肌中Cx43、c-FosmRNA及蛋白的表达水平。【结果】Cx43、c-Fos mRNA及蛋白表达水平在足月妊娠子宫肌中显著高于其在非孕期正常子宫肌中,差异有显著性（P〈0.05）;临产组子宫肌中Cx43c、-Fos mRNA及其蛋白的表达水平显著高于未临产组（P〈0.05）,且Cx43 mRNA及其蛋白表达水平与c-Fos mRNA及其蛋白表达水平呈正相关（相关系数分别为rs=0.580,0.602）。【结论】子宫平滑肌中间隙连接蛋白Cx43、c-Fos的表达水平与分娩发动密切相关,并且可能在分娩发动中起重要作用。     

Potassium balance and the control of renin secretion

Plasma renin activity and renin substrate were measured in nine groups of rats which were maintained for 7 wk on diets in which the proportions of sodium and potassium were varied.Balance data indicated that the highest dietary intake of potassium employed (92 mEq K(+)/100 g food) consistently induced sodium depletion. With less consistency, the highest sodium intake employed (52 mEq Na(+)/100 g food) tended to induce potassium depletion.In accordance with previous reports, sodium deprivation induced significant increases in plasma renin activity. But the present results indicated that changes in potassium intake exerted a highly significant modulating influence on this characteristic response. The results describe an inverse relationship between potassium administration and the concurrent level of plasma renin activity. The highest serum renin levels of all occurred in the potassium-depleted animals and the usual renin response to sodium deprivation was virtually abolished in the presence of a high potassium diet.Neither the suppressing effect of K(+) administration nor the stimulating effect of K(+) depletion on plasma renin activity could be explained in terms of any predicted changes in aldosterone secretion or observed changes in sodium balance. Therefore, the effect seems to be mediated by a direct influence of potassium ions on renal renin secretion, perhaps via induced changes in sodium load to the macula densa.These studies point to an important role for potassium in the regulation of renin secretion. The results in turn raise the possibility that renin secretion per se may be importantly involved in effecting potassium conservation and potassium elimination. The means by which these interactions are finally mediated remain to be clarified.     

Nitric oxide and the control of renin secretion

Summary— Research during recent years has established nitric oxide as a unique signaling molecule that plays important roles in the regulation of the cardiovascular, nervous, renal, immune and other systems. Nitric oxide has also been implicated in the control of the secretion of hormones by the pancreas, hypothalamus, pituitary and other endocrine glands, and evidence is accumulating that it contributes to the regulation of the secretion of renin by the kidneys. The enzyme nitric oxide synthetase is present in vascular and tubular elements of the kidney, particularly in cells of the macula densa , a structure that plays an important role in the control of renin secretion. Guanylyl cyclase, a major target for nitric oxide, is also present in the kidney and is responsive to changes in nitric oxide levels. Drugs that inhibit nitric oxide synthesis generally suppress renin release in vivo and in vitro , suggesting a stimulatory role for the L-arginine-nitric oxide pathway in the control of renin secretion. Under some conditions, however, blockade of nitric oxide synthesis increases renin secretion. Recent studies indicate that nitric oxide not only contributes to the regulation of basal renin secretion, but also participates in the renin secretory responses to activation of the renal baroreceptor, macula densa and beta adrenoceptor mechanisms that regulate renin secretion. Future research should clarify the mechanisms by which nitric oxide regulates the secretion of renin and establish the physiological significance of this regulation.     

The renin gene in patients with malignant hypertension and raised plasma renin activity

1. We have examined the hypothesis that the raised plasma renin activity in patients with malignant hypertension without an underlying cause is the consequence of expression of a duplicate renin gene. 2. DNA extracted from leucocytes of patients with malignant hypertension and of normotensive controls was digested with the restriction endonuclease PstI and hybridized with a radioactively labelled human renin complementary DNA probe. As an internal control the DNA was concurrently hybridized with a human c-myc protooncogene probe. 3. The signals for each subject from the two probes were quantitatively compared by densitometry. 4. There was no evidence of duplication of the renin gene in the patients with malignant hypertension.     

Plasma renin activity in essential hypertension

Summary A study of the frequency distribution of plasma renin activity (PRA) in 123 patients with essential hypertension (EH) produced no evidence of a distinct subpopulation with low renin levels, whether the samples were taken from supine or upright patients. Applying an arbitraty classification criterion, however, low PRA levels were found in 30.1% of patients. There were no significant differences in mean blood pressure, 24-h sodium excretion, and age when groups with low, normal or high PRA levels were compared. The incidence of PRA hyporesponsiveness was similar in the three groups of patients, but increased with age. In the female there was a preponderance of low PRA levels. It is concluded that EH with low PRA levels is not a separate diagnostic entity and, when PRA is low in a hypertensive subject, the possible effects of age, blood pressure, and sex ought to be taken into account before other causes of low PRA are postulated.     

Role of cGMP-kinase II in the control of renin secretion and renin expression.

To investigate the roles of the cGMP-dependent protein kinases (cGKs) in the control of the renin system, we studied the regulation of renin in cGKI- or cGKII-deficient mice in vivo and in vitro. Renal renin mRNA levels both under stimulatory (low-salt diet plus ramipril) and inhibitory (high-salt diet) conditions were not different between wild-type and cGKI-/- mice, but were significantly elevated in cGKII-/- mice under all experimental conditions. In primary cultures of renal juxtaglomerular cells (JG) established from wild-type, cGKI-/-, and cGKII-/- mice, the adenylate cyclase activator forskolin stimulated renin secretion similarly in all genotypes tested. 8-bromo-cGMP attenuated basal and forskolin-stimulated renin secretion in cultures from wild-type and cGKI-/-, but had no effect in cells isolated from cGKII-/- mice. Activation of cGKs by 8-bromo-cGMP decreased renin secretion from the isolated perfused rat kidney, independent of prestimulation by beta-adrenoreceptor activation, macula densa inhibition, reduced perfusion pressure, or by a nominally calcium-free perfusate. Taken together, these findings suggest that activation of cGKII has a general inhibitory effect on renin secretion from renal JG cells.     

Parasympathetic pathways, renin secretion and vasopressin release.

1. The interrelationship between parasympathetic neural tone, renin secretion and vasopressin release was examined by observing the effect of bilateral cervical vagotomy on renin secretion in intact and acutely hypophysectomized dogs undergoing a water diuresis. 2. In intact dogs bilateral cervical vagotomy decreased the mean renin secretion from 1245 to 682 units/min (P less than 0.01) as urinary osmolality increased from 95 to 414 mosmol/kg (P less than 0.001). In contrast, in acutely hypophysectomized dogs cervical vagotomy failed to alter renin secretion significantly (834 to 893 units/min) and urinary osmolality was also unchanged (78 to 71 mosmol/kg). 3. The results suggest that a diminution in vagal tone may significantly alter renin secretion by stimulating vasopressin release. Exogenous vasopressin was associated with changes in urinary osmolality and renin secretion which were qualitatively similar to those seen after servical vagotomy. 4. We suggest that there is a neurohumoral reflex mechanism by which a fall in parasympathetic tone increases the release of vasopressin, which, in turn, suppresses renin secretion. The results are also compatible with the hypothesis that vasopressin inhibits renin release by a direct effect on the juxtaglomerular cells.