Induction of smooth muscle alpha-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase.

Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle alpha-actin (SM-alpha-actin) expression through activation of the SM- alpha-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-alpha-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-alpha-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVP-stimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely super-shifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM- alpha-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases.

Galpha16 mimics vasoconstrictor action to induce smooth muscle alpha-actin in vascular smooth muscle cells through a Jun-NH2-terminal kinase-dependent pathway.

Prolonged exposure of vascular smooth muscle cells (VSMC) to vasoconstrictors such as vasopressin or angiotensin II induces hypertrophy and increases expression of muscle-specific genes including smooth muscle alpha-actin (SM-alpha-actin). These vasoconstrictors signal through G-proteins, including members of the Gq family. To further investigate the role of Gq family members, VSMC were transfected with a constitutively active mutant of a Gq family member, Galpha16 (Galpha16Q212L). Stable expression of Galpha16Q212L persistently stimulated phospholipase C, resulting in increased basal levels of inositol phosphates. These cells were hypertrophied and expressed elevated levels of SM-alpha-actin compared with wild-type VSMC or cells transfected with a control plasmid (Neo). SM-alpha-actin promoter activity was markedly increased in cells stably or transiently expressing Galpha16Q212L. Basal c-Jun-NH2-terminal kinase (JNK) activity was increased 3-9-fold in cells stably expressing Galpha16Q212L, while basal activity of the p42/44 mitogen-activated protein kinases (ERKs) was unaffected. Transient expression of a kinase inactive JNK kinase partially inhibited induction of SM-alpha-actin promoter activity in response to vasoconstrictors or expression of Galpha16Q212L. These results indicate that expression of constitutively active Galpha16 in VSMC mimics the effects of vasoconstrictors on hypertrophy and muscle-specific gene expression, and activation of JNK may play a role in these responses.

Suppression of smooth-muscle alpha-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2.

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.

Activation of a novel form of phospholipase A2 during liver regeneration.

Activation of phospholipase A2 (PLA2) occurs following mitogenic stimulation of cells. This study examined PLA2 activation during liver regeneration. Increased activity was detected within 1 h after partial hepatectomy, was maximal by 6 h, and returned to control levels by 24 h. Fractionation of cell-free extracts revealed multiple peaks of PLA2 activity. One peak appeared identical to the previously described cPLA2, and was modestly stimulated during regeneration. A higher molecular weight form (hPLA2) was stimulated approximately 5-fold during regeneration. This enzyme was Ca(2+)-dependent and selective for arachidonoylphosphatidylethanolamine. The activation of this novel form of PLA2 represents an early event in liver regeneration, and is likely to contribute to the proliferative response.

Regulation of smooth muscle alpha-actin promoter by vasopressin and platelet-derived growth factor in rat aortic vascular smooth muscle cells.

Vasoconstrictors such as arginine vasopressin (AVP) and angiotensin II (Ang II) have been shown to increase protein and mRNA levels of smooth muscle alpha-actin (SM-alpha-actin) in vascular smooth muscle cells. In the same cells, platelet-derived growth factor (PDGF) decreased SM-alpha-actin protein and mRNA. The rat SM-alpha-actin promoter that has recently been isolated contains two E-boxes and three CC(A/T)6GG (CArG) elements. To examine regulation of the SM-alpha-actin promoter, a 765-bp region of the rat SM-alpha-actin gene was ligated into chloramphenicol acetyltransferase (CAT)-containing vectors and transfected into rat aortic vascular smooth muscle cells. Stimulation of cells with either AVP or Ang II increased CAT activity 5- to 10-fold. PDGF was able to completely block the AVP-induced increase in CAT activity. To identify regions of the promoter responsible for both the AVP stimulation and PDGF inhibition of promoter activity, a series of truncation mutants were prepared and transfected into vascular smooth muscle cells. Truncation of both E-boxes and the most distal CArG element did not qualitatively alter either AVP-induced stimulation of CAT activity or PDGF inhibition. However, removal of the middle CArG element resulted in a loss of AVP stimulation. These studies indicate that the AVP-induced elevation and PDGF-induced inhibition of SM-alpha-actin levels in vascular smooth muscle cells are mediated at least in part through regulation of the SM-alpha-actin promoter. The critical region of the promoter mediating this effect involves at a minimum one of the CArG elements.

Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase.

Vascular smooth muscle cells have been shown to exist in two phenotypic states which have been designated proliferative and contractile. The properties of rat aortic vascular smooth muscle cells grown on Matrigel were compared with cells grown on untreated plastic culture dishes. Cells grown on Matrigel manifested at least four important properties characteristic of the contractile phenotype as compared with cells grown on plastic. The cells grown on Matrigel had altered morphology similar to in vivo studies of contractile vascular smooth muscle. The cells had a low proliferative index, showed enhanced levels of the smooth muscle isoform of alpha-actin, and had an enhanced contractile response to the vasoconstrictor arginine vasopressin. All of these changes were maintained through at least five passages and could be reversed by replating cells grown on Matrigel back to uncoated plastic dishes. Changes in post-receptor signaling pathways which could account for the altered physiologic responses of the cells were investigated. Cells grown on Matrigel showed no alterations in agonist-induced mobilization of intracellular Ca2+ or agonist-stimulated cAMP levels. However, stimulation of mitogen-activated protein kinase (MAP kinase) by both vasoconstrictors and growth factors was inhibited by 50% in cells grown on Matrigel as compared with plastic. This decrease in agonist-induced MAP kinase was associated with a decrease in the levels of both p42 and p44 MAP kinase protein and a decrease in tyrosine phosphorylation of both isoforms in cells grown on Matrigel. Alterations in MAP kinase activation can account at least in part for the observed physiologic responses of contractile vascular smooth muscle. Growth of vascular smooth muscle cells on Matrigel represents a useful model to examine phenotypic-dependent alterations in post-receptor signaling.

AVP-induced activation of MAP kinase in vascular smooth muscle cells is mediated through protein kinase C.

Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of p42 mitogen-activated protein (MAP) kinase (p42MAPK) in vascular smooth muscle cells (VSMC). In VSMC, AVP increases free intracellular Ca2+ concentration ([Ca2+]i) and activates protein kinase C (PKC) through activation of phospholipase C. The contribution of PKC and [Ca2+]i in p42MAPK regulation was therefore determined. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) stimulated tyrosine phosphorylation and activation of p42MAPK to the same extent as AVP. Inhibition of PKC by staurosporine or downregulation of PKC by PMA pretreatment abolished AVP-induced stimulation of p42MAPK. When [Ca2+]i was elevated to the same level as with AVP, using either ionomycin (0.1 microM) or thapsigargin (0.1 microM), MAP kinase was only partially activated. Elevation of [Ca2+]i to supraphysiological levels by 1 microM ionomycin stimulated MAP kinase activity to the same extent as AVP. This effect was blocked by downregulation of PKC. The intracellular Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked AVP-induced [Ca2+]i increase but did not affect AVP stimulation of p42MAPK. Thus AVP-induced activation of p42MAPK requires only the activation of PKC but not an increase in [Ca2+]i.

Direct evidence for tyrosine and threonine phosphorylation and activation of mitogen-activated protein kinase by vasopressin in cultured rat vascular smooth muscle cells.

Mitogen-activated protein (MAP) kinases are members of a 40-45-kDa family of serine/threonine protein kinases that phosphorylate several substrates including microtubule-associated protein-2, S6 kinase, and myelin basic protein. Activity of MAP kinases is regulated by growth factors that stimulate the phosphorylation of threonine 188 and tyrosine 190 in the kinase. In this paper direct evidence is presented for tyrosine and threonine phosphorylation of MAP kinase in concert with elevated activity in response to vasopressin in primary cultures of vascular smooth muscle cells. Activation of MAP kinase is correlated with activation of S6 kinase activity related to S6 kinase II. Data support the concept that the activation of MAP kinase by vasopressin is mediated by pertussis toxin-independent biochemical pathways.

Increased norepinephrine secretion in patients with the nephrotic syndrome and normal glomerular filtration rates: evidence for primary sympathetic activation.

Considerable controversy exists in regard to the state of arterial circulatory integrity in patients with the nephrotic syndrome. Increased sympathetic nervous system activity, along with activation of the renin-angiotensin-aldosterone system and the nonosmotic release of vasopressin, is seen in other states of arterial underfilling. Thus, in the present study, sympathetic nervous system activity was assessed by determining plasma norepinephrine secretion and clearance rates using a whole-body steady-state radionuclide tracer method in 6 edematous patients with the nephrotic syndrome of various parenchymal etiologies and 6 normal control subjects in the supine position. Patients were withdrawn from all medications 7 days prior to study. Mean creatinine clearances and serum creatinine concentrations were normal in both the nephrotic syndrome patients and controls (99 +/- 13 vs. 112 +/- 15 ml/min, p = NS, 1.1 +/- 0.1 vs. 0.8 +/- 0.0 mg/dl, p = 0.03, respectively). However, the nephrotic syndrome patients exhibited significant hypoalbuminemia (2.0 +/- 0.4 vs. 3.8 +/- 0.1 g/dl, p < 0.01). The supine plasma norepinephrine level was elevated in the patients with the nephrotic syndrome as compared with controls (240 +/- 58 vs. 119 +/- 22 pg/ml, p = 0.07). More significantly, the secretion rate of norepinephrine was markedly increased in nephrotic patients (0.30 +/- 0.07 vs. 0.13 +/- 0.02 micrograms/m2/min, p < 0.05), whereas the clearance rate of norepinephrine was similar in the two groups (2.60 +/- 0.29 vs. 2.26 +/- 0.27 l/min, p = NS). Plasma renin activity and plasma aldosterone, arginine vasopressin and atrial natriuretic peptide concentrations were not different in nephrotic syndrome patients compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C.

Phospholipase A2 (PLA2) is the enzyme regulating the release of arachidonic acid in most cell types. A high molecular mass, 85-kDa soluble form of PLA2 (cPLA2) has recently been identified, the activity of which is stably increased by stimulation of cells with hormones and growth factors. Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA2 on serine residues, but the kinases mediating this effect have not been identified. We report here that human cPLA2 is phosphorylated in vitro by two growth factor-stimulated serine/threonine-specific kinases, p42 MAP kinase and protein kinase C (PKC). Phosphorylation of the cPLA2 enzyme by either kinase results in an increase in catalytic cPLA2-specific activity. Domains of the cPLA2 molecule have been expressed in Escherichia coli, and the fusion proteins purified. PKC and p42 MAP kinase give different patterns of phosphorylation of the recombinantly expressed cPLA2 fragments. p42 MAP kinase selectively phosphorylates the domain of cPLA2 containing a MAP kinase consensus sequence, whereas PKC phosphorylates sites in all three recombinantly expressed domains of the enzyme. Peptide mapping indicates that the site phosphorylated by p42 MAP kinase is different from those phosphorylated by PKC. The combined action of both of these kinases is likely to mediate the effects of growth factor stimulation on arachidonic acid release through the activation of cPLA2.

Intra- and extracellular proteins in human normal and polycystic kidney epithelial cells.

A tissue culture method was established for the continuous growth of epithelial cells from the cortex of human normal kidney (HNC) and from the epithelial layer of kidney cysts from autosomal dominant polycystic kidney disease (ADPKD) patients. Primary cells were grown to 80 to 90% confluency from 1 mm2 slices of tissue, and subcultured up to 10 times. The subcultured HNC and ADPKD cells retained characteristic epithelial polygonal and elongated shape and positive immunofluorescent staining for cytokeratin. The cell doubling time for both HNC and ADPKD epithelia was three to four days at a fetal calf serum (FCS) concentration of 5%. Using these culturing procedures 1 to 5 x 10(9) epithelial cells could be obtained from each kidney specimen. Profiles of 35S-methionine radiolabeled intracellular proteins of HNC and ADPKD cells qualitatively demonstrated a high degree of similarity, thus confirming a similarity of epithelial origin and protein biosynthesis. Both the underexpression of three proteins (a) protein p2, Mr approximately 47 kDa, pI approximately 6.0; b) protein p3, Mr approximately 50 kDa, pI approximately 5.9; and c) protein p4, Mr approximately 44 kDa, pI approximately 5.8) and the overexpression of several proteins (including: a) p5, Mr approximately 56 kDa, pI approximately 7.3; b) protein p6, Mr approximately 32 kDa, pI approximately 7.3; c) protein p7, Mr approximately 33 kDa, pI approximately 5.3; d) protein p8, Mr approximately 45 kDa, pI approximately 6.9; e) protein p9, Mr approximately 35 kDa, pI approximately 6.7; and f) protein p10, Mr approximately 30 kDa, pI approximately 6.6) were found in ADPKD cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin dependent tyrosine phosphorylation of a 38 kDa protein in human platelets.

Arginine vasopressin administration (10(-10)-10(-6) M) to isolated human platelets induces an increase in the specific immunoblotting of a 38 kDa protein revealed by a phosphotyrosine antibody. This signal is biphasic with maximal stimulation within one minute. Neither forskolin (10(-5) M) nor phorbol ester (10(-6) M) produces a similar 38 kDa signal. The specific immunoblotted signals are competitively abolished by 1 mM phosphotyrosine but not phosphoserine or phosphothreonine. Electrophoretic separation at pH 3.5 of the acid hydrolysates of the 38 kDa proteins reveals a vasopressin dependent increase in levels of phosphotyrosine as well as phosphoserine and phosphothreonine. The 38 kDa phosphorylation is also induced by the specific arginine vasopressin V1 receptor agonist (Phe2Orn8Vastocina) and blocked by the V1 receptor antagonist [desGly(NH2)d(CH2)5Tyr(Me) AVPb]. These observations suggest that arginine vasopressin signal transduction may be associated with the tyrosine phosphorylation of a 38 kDa protein.

Effect of extracellular acidosis on 45Ca uptake in isolated hypoxic proximal tubules.

Recent in vitro studies have suggested that the presence of extracellular acidosis is protective against the development of oxygen-deprivation injury in several tissues. Because cellular Ca accumulation after renal ischemia may represent a major pathogenic event leading to cellular damage, the purpose of the present study was to examine the effect of extracellular acidosis on 45Ca uptake and desaturation in normal and hypoxic isolated rat proximal tubules. At pH 7.4, an increase in 45Ca uptake was observed in proximal tubules after 30 min of hypoxia. In addition, 45Ca desaturation was increased significantly in hypoxic tubules at pH 7.4. The alterations in 45Ca uptake and desaturation in hypoxic tubules at pH 7.4 were accompanied by significant signs of cellular injury as assessed by the amount of lactate dehydrogenase (LDH) released by the tubules at the end of the hypoxic period (22.5% above control tubules, P less than 0.01) as well as in morphological changes consistent with hypoxic cell injury. In contrast, when maintained at pH 6.9 throughout the study, no difference in 45Ca uptake or desaturation was observed between the control and hypoxic tubules; the hypoxic proximal tubules exhibited a smaller increase in LDH release (12.7% above control tubules) and did not develop the morphological changes observed at pH 7.4. Thus, during hypoxia and reoxygenation at pH 7.4, the increased 45Ca uptake may contribute in part to cellular injury in rat proximal tubules.

Adenine nucleotide metabolism and mitochondrial Ca2+ transport following renal ischemia.

Mitochondrial Ca2+ accumulation and mitochondrial respiratory dysfunction have been observed after renal ischemia. The present study examined the effects of ischemia and reperfusion on cellular energy production and mitochondrial Ca2+ transport after 50 min of bilateral renal artery and vein clamping in the anesthetized rat. Prior to reflow, tissue ATP and total adenine nucleotide levels were severely reduced. These nucleotide levels recovered towards normal but remained lower than control values throughout 24 h of reperfusion. Energy-linked mitochondrial Ca2+ uptake was unmeasurable, mitochondrial Ca2+ efflux was increased and the mitochondria were unable to maintain a steady-state free Ca2+ concentration prior to reflow. Three hours of reperfusion was associated with a normalization of mitochondrial Ca2+ uptake, release, and steady-state buffering. However, progressive deterioration subsequently occurred in these processes. Thus the mitochondrial Ca2+ accumulation previously observed during the later stages of postischemic reperfusion (3-24 h) is due neither to an increase in the rate of active Ca2+ uptake nor to a decrease in the rate of Ca2+ release. The present results therefore suggest that passive mitochondrial Ca2+ accumulation may occur during the later stages of reperfusion, probably due to a progressive increase in the cytosolic Ca2+ concentration.

Elevated plasma norepinephrine concentrations in decompensated cirrhosis. Association with increased secretion rates, normal clearance rates, and suppressibility by central blood volume expansion.

Plasma norepinephrine concentrations are elevated in patients with decompensated cirrhosis, and correlate inversely with urinary sodium and water excretion. Increased plasma norepinephrine concentrations may result from a decreased metabolic clearance rate or an increased secretion rate, possibly in response to a decreased "effective arterial blood volume." If the latter hypothesis is correct, plasma norepinephrine might be expected to be suppressed when central blood volume is expanded by head-out water immersion. In the present study, plasma norepinephrine secretion and clearance rates were determined by infusion of tritiated norepinephrine. Norepinephrine secretion rates were elevated in eight cirrhotic patients as compared to control subjects (1.50 +/- 0.25 vs. 0.26 +/- 0.08 micrograms/m2 per min, P less than 0.001), whereas clearance rates were similar (3.13 +/- 0.48 vs. 2.60 +/- 0.28 liters/min, NS). Baseline plasma norepinephrine concentrations were markedly elevated in the cirrhotic patients (830 +/- 136 vs. 185 +/- 12 pg/ml, P less than 0.001). Head-out water immersion significantly suppressed plasma concentrations of both norepinephrine (704 +/- 72 to 475 +/- 70 pg/ml, P less than 0.005) and epinephrine (121 +/- 33 to 57 +/- 10 pg/ml, P less than 0.05) in all seven patients studied. We conclude that the high circulating catecholamine concentrations in cirrhosis are secondary to increased secretion, rather than to decreased metabolic clearance, and are suppressible by central blood volume expansion.

Potassium-aldosterone response in dogs with chronic renal insufficiency.

To evaluate the potassium-aldosterone axis in chronic renal insufficiency, the plasma concentration of aldosterone was measured before and after a 50-mEq potassium challenge in dogs with intact kidneys (glomerular filtration rate 52 +/- 3.1 ml/min) and in dogs with one remnant kidney (glomerular filtration rate 16.1 +/- 2.1 ml/min). Fasting concentrations of circulating potassium and aldosterone and urinary potassium excretion rates were similar in both groups. In the 5 h following potassium chloride administration, less of the potassium load was excreted by dogs with one remnant kidney (30.2 +/- 3.3%) than by normal dogs (55.6 +/- 7.3%; p less than 0.01); serum potassium rose significantly more in dogs with chronic renal insufficiency than in normal animals. After potassium chloride, plasma aldosterone increased in both groups, but the increments in dogs with one remnant kidney were twice the increases in normal dogs (p less than 0.01). The increases in plasma aldosterone per unit increment in serum potassium, however, were similar in both groups (14.5 vs. 13.8 ng X dl-1/mEq X l-1). Thus, the hyperkalemia that follows a potassium load in dogs with chronic renal insufficiency is not due to a failure to secrete aldosterone but to a decrease in potassium excretion in the setting of decreased renal mass.

Potential role of increased sympathetic activity in impaired sodium and water excretion in cirrhosis.

In a study of 26 patients with cirrhosis, plasma norepinephrine concentrations were significantly higher in 19 patients who abnormally excreted an acute water load than in seven who excreted the load normally (8324 +/- 116 vs. 306 +/- 33 pg per milliliter; P less than 0.001). There was also a significant positive correlation between plasma levels of norepinephrine and arginine vasopressin after the water load, as well as a negative correlation between plasma norepinephrine and the percentage of the load excreted. A positive correlation between plasma norepinephrine and plasma renin activity, as well as between norepinephrine and aldosterone, was observed. In addition, there was a negative correlation between plasma norepinephrine and urinary sodium excretion. These findings indicate that increased sympathetic activity, as assessed by plasma levels of norepinephrine, correlates closely with sodium and water retention in cirrhotic patients and thus may be of pathogenetic importance.