The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.

We determined the relationship between the activation state and phosphorylation state of the Na-K-Cl cotransport protein in tubules isolated from the shark rectal gland, a prototypic chloride-secreting epithelium. In response to cAMP-dependent secretagogues (e.g. vasoactive intestinal peptide, adenosine, and forskolin) or osmotically induced changes in cell volume, the activation state of the cotransport protein (assessed from measurements of loop diuretic binding) increased 5-10 fold. The response was temporally associated with a comparable increase (3-9 fold) in cotransport protein phosphorylation. Graded changes in cotransporter activation evoked proportional changes in cotransporter phosphorylation. Under the conditions of our experiments, the 195-kDa cotransporter was the only membrane protein whose phosphorylation state increased conspicuously in response to both cAMP and cell shrinkage. Both stimuli promoted phosphorylation of the cotransport protein at serine and threonine residues. One of the cAMP-sensitive phosphoacceptors was found within a segment of the cotransport protein comprised of a sequence (Phe-Gly-His-Asn-Thr*-Ile-Asp-Ala-Val-Pro) that corresponds to a segment of the Na-K-Cl cotransport protein predicted by cDNA analysis, where the phosphoacceptor (Thr*) is threonine 189. Incubation of rectal gland tubules with K-252a or H-8, structurally different protein kinase inhibitors, rendered the cotransporter insensitive to both cAMP and cell shrinkage. We conclude that the rectal gland Na-K-Cl cotransport protein is regulated by direct reversible phosphorylation at serine and threonine sites.     

3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters

Vascular endothelial cells previously have been shown to possess a prominent Na-K-Cl cotransport system which mediates a K+ influx of approximately 20 mumols/g of protein/min. Endothelial cell cotransport has also been shown to be regulated by a variety of vasoactive agents and their second messengers, suggesting that the transport system may have an important role in endothelial cell function. In the present study we investigated the possibility that the high level of cotransport in these cells is due to a large number of Na-K-Cl cotransporters in the plasma membrane. This was done by evaluating specific saturable binding of [3H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal [3H]bumetanide binding of 0.83 pmol/mg protein with a dissociation constant of 0.13 microM. From these data, the number of [3H]bumetanide binding sites/endothelial cell was determined to be approximately 230,000, and the turnover number for cotransport activity was calculated to be 300 K+ ions/site/s. These findings indicate that endothelial cells do indeed exhibit a large number of Na-K-Cl cotransporters/cell relative to other cell types. We also investigated the effects on [3H]bumetanide binding of agents known to modulate Na-K-Cl cotransport activity. Saturable binding of [3H]bumetanide was found to be reduced significantly by treatment of the cells with 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol esters, norepinephrine, or rat atriopeptin III, all of which have been shown to inhibit Na-K-Cl cotransport-mediated K+ influx.     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na：1K：2C1（乌贼轴突中是2Na：1K：3C1）,其活性被布美他尼（bumetanide）和呋塞米（furosemide）所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体：NKCCl和NKCC2。NKCCl存在于多个组织中,合有NKCCl的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密斑的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用（尤其是NKCCl亚基）是通过特定的激酶使该转运蛋白自身发生氧化／硝化、磷酸化／去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.     

Agonist stimulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways

Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Cl- cotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Cl- cotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Cl- cotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Cl- cotransport by angiotensin II and alpha-thrombin is mediated by protein kinase C and Ca2+/calmodulin-dependent kinases whereas the action of EGF is mediated by other pathways.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Arginine vasopressin stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransporter activity is V1 receptor and [Ca] dependent

Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally located BBB Na-K-Cl cotransporter is stimulated during cerebral ischemia to increase transport of Na and Cl into the brain. The main focus of the present study was to evaluate the effects of arginine vasopressin (AVP), previously shown to be increased in the brain during ischemia and to promote edema formation, on activity of the BBB cotransporter. Cerebral microvascular endothelial cell (CMEC) monolayers were cultured in astroglial cell conditioned medium, and Na-K-Cl cotransporter activity was assessed as bumetanide-sensitive ⁸⁶Rb influx. In both human and bovine CMECs, as well as in freshly isolated microvessels, AVP stimulated cotransport activity. This stimulatory effect was mimicked by V₁ but not V₂ vasopressin agonists and was blocked by V₁ but not V₂ vasopressin antagonists. Consistent with a V₁ vasopressin receptor mechanism of action, AVP caused an increase in CMEC intracellular [Ca] that was blocked by a V₁ antagonist. Exposing the cells to [Ca]-free media and/or reducing intracellular [Ca] by BAPTA also blocked AVP stimulation of CMEC cotransporter activity, as did the phospholipase C inhibitor U-73122. Finally, we found that while stimulation of CMEC cotransporter activity by AVP occurred within minutes, it was also sustained for hours in the continued presence of AVP. These findings support the hypothesis that AVP, through a V₁ receptor- and [Ca]-dependent mechanism, stimulates the BBB Na-K-Cl cotransporter to participate in ischemia-induced edema formation. blood-brain barrier; stroke; cerebral ischemia; brain edema     

Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (alpha-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 microM ATP or 100 microM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.     

Phorbol esters, vasopressin and angiotensin II block alpha 1-adrenergic action in rat hepatocytes. Possible role of protein kinase C

The effect of vasopressin, angiotensin II and phorbol myristate acetate on the alpha 1-adrenergic action (induced by epinephrine + propranolol), was studied. We selected three conditions: (a) ureagenesis in medium without added calcium and containing 25 microM EGTA; (b) ureagenesis using cells from hypothyroid animals, and (c) gluconeogenesis from dihydroxyacetone. Under these conditions epinephrine + propranolol produces clear metabolic effects, whereas the vasopressor peptides do not (although they stimulate phosphoinositide turnover). It was observed that the vasopressor peptides and the active phorbol ester inhibited in a concentration-dependent fashion the effect of epinephrine + propranolol. It is suggested that activation of protein kinase C by phorbol esters or physiological stimuli (hormones that activate phosphoinositide turnover, such as vasopressin or angiotensin II) modulate the hepatocyte alpha 1-adrenergic responsiveness.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Prostaglandin D2 in cultured capillary and microvascular endothelium of human brain.

Production of prostaglandin D2 (PGD2) was investigated in cultured endothelial cells derived from capillaries and microvessels (small and large) of human brain using radioimmunoassays. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester and calcium ionophore greatly stimulated the secretion of endothelial PGD2. Secretion of PGD2 induced by vasoconstricting peptides, angiotensin II and arginine-vasopressin, was almost completely abolished by their respective specific receptor antagonists [Sar1, Ala8]-Ang II and [1-6(beta-mercapto-beta,beta-cyclopentamethylene propionic acid) 2-O-methyltyrosine]. Thus, the augmented production of PGD2 by angiotensin II and arginine-vasopressin is a receptor-mediated event. It also indicates that the EC have specific angiotensin II and arginine-vasopressin (V1) receptors. This study represents the first demonstration of vasoactive agents modulating PGD2 production in capillary and microvascular endothelium of human brain.     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Effect of vasoactive peptides on prostacyclin formation in cerebromicrovascular cellular elements and glia: a comparative study

The aim of the present study was to determine basal and stimulated release of prostacyclin from the separately cultured endothelial and smooth muscle cells derived from rat brain microvessels and from glial cells.The basal release of PGI₂ (measured as a 6-keto-PGF_1α formation by radioimmunoassay method) was significantly greater in cultured endothelial cells than in cultured smooth muscle or glial cells (254 ± 32, 140.7 ± 17 and 76.8 ± 5.8 pg/mg protein, respectively). Prostacyclin formation stimulated by angiotensin I, angiotensin II and bradykinin was significantly increased in the smooth muscle cells. A significant enhancement of PGI₂ formation was also observed in the glial cells exposed to angiotensin II or bradykinin. Vasoactive peptides did not affect prostacyclin production in the endothelial cells.Presented results indicate that the smooth muscle cells represent the most sensitive site of prostacyclinpeptide interaction. These data also suggest that the endothelial and the glial cells may protect the cerebromicrovascular smooth muscle by inactivating vasoactive peptides derived from either the blood or the brain.     

Stimulation of Na-K-Cl cotransport in cultured vascular endothelial cells by atrial natriuretic peptide

Vascular endothelial cells have been shown to contain atrial natriuretic peptide (ANP)-sensitive Na-K-Cl cotransport system whose activity is regulated by intracellular cGMP levels. Addition of ANP to culture medium stimulated 86Rb+ uptake in bovine endothelial cells with a concomitant increase in cGMP contents. This action of ANP was mimicked by 8-bromo-cGMP and completely diminished by furosemide. These results indicate that ANP selectively activates the Na-K-Cl cotransporter in vascular endothelial cells via cGMP and offer new insight into the physiological significance of endothelial ANP receptors.     

Phorbol Ester-Induced Upregulation of Angiotensin II Receptors in Neuronal Cultures Is Potentiated by a Calcium Ionophore

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.     

Homologous and heterologous desensitization of one of the pathways of the alpha 1-adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the alpha 1-adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the alpha 1-adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards alpha 1-adrenergic agonists.     

Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in swiss 3T3 cells

Vasopressin and bradykinin bind to receptors coupled to GTP-binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca²⁺ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vaso-pressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long-term activation of protein kinase C, like vasopression or phorbol 12, 13-dibutyrate. Down-regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long-term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of M_r = 110,000–130,000 and M_r = 70,000–80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca²⁺ -mobilizing peptides on mitogenesis may be more general than previously thought. © 1994 Wiley-Liss, Inc.     

Cerebral endothelial cell culture. I. The presence of beta 2 and alpha 2-adrenergic receptors linked to adenylate cyclase activity

Cultured endothelial cells derived from cerebral microvessels separated from 2-day-old rat brain contain a specific beta 2 and alpha 2-adrenergic sensitive adenylate cyclase (AC). Among the various tested hormones, PGE1 and PGE2 were found to be the most potent activators, while adenosine, angiotensin I and II, gamma-aminobutyric acid and vasoactive intestinal peptide inhibited the enzyme activity. However, acetylcholine, histamine, serotonin, glycine, glutamine, bradykinin, neurotensin and vasopressin (Lysine and Arginine) had no effect on the adenylate cyclase activity in this model. The susceptibility of the cerebrovascular endothelial AC system to the vasoactive substances as well as presence of beta 2 and alpha 2-type adrenergic receptors in the cultured endothelium provides additional support for the proposed endothelial involvement in the regulation of cerebrovascular permeability and blood flow.     

The Epithelial Phenotype of Human Neuroblastoma Cells Expresses Bradykinin, Endothelin, and Angiotensin II Receptors That Stimulate Phosphoinositide Hydrolysis

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.     

Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay

High-affinity binding sites for endothelin have been found in a human placenta membrane preparation. 125I-endothelin bound to placenta membranes at 20 degrees C with an association half-time of 30 min, whereas the binding was only slowly reversed with a dissociation half-time of 250 min. In saturation experiments, a single class of high-affinity binding sites was identified with an apparent dissociation constant (KD) of 24 pM and a maximal density of 240 fmol per mg of protein. The binding of 125I-endothelin was half-maximally inhibited by cold endothelin at a concentration (IC50) of 140 pM. In contrast, no inhibition was found at 10(-4) M for a variety of vasoactive peptides such as angiotensin II, vasopressin, neuropeptide Y, substance P, CGRP, bradykinin, leucine enkephalin or dynorphin A. Similarly, the binding was modulated neither by the calcium channel blockers nifedipine, verapamil or diltiazem, nor by the calcium channel agonist Bay k 8644. There was also no effect with the structurally-related bee venom apamin. Using this membrane preparation, endothelin-like activity could be measured in the medium of cultured human endothelial cells by competition binding technique.