T-cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivo.

BACKGROUND: Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol-anchored T-cadherin (T-cad) is highly expressed in the basal keratinocyte layer of skin. The role of T-cad in keratinocyte biology and pathology is unclear. OBJECTIVES: To define the role of T-cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain-of-function and loss-of-function studies in vitro and through examination of T-cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. METHODS: In vitro studies employed lentiviral-mediated overexpression/silencing of T-cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. RESULTS: In vitro, silencing of T-cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T-cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T-cad expression was more frequent and prominent in SCC classified as moderately-to-poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T-cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. CONCLUSIONS: T-cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC.

Endothelin messenger RNA and receptors are differentially expressed in cultured human breast epithelial and stromal cells.

Paracrine regulation is implicit in the biosynthesis and secretion of milk in the breast. An important determinant for this regulation in vivo is proximate cellular location as exemplified by stromal and epithelial cells in breast tissue. Cultured human breast epithelial cells exhibited low constitutive expression of mRNA for endothelin which was enhanced 20-fold after prolactin stimulation. Human breast stromal cells did not express measurable levels of endothelin mRNA under similar conditions. In a similar differential manner, the stimulated release of immunoreactive endothelin into medium overlay was observed only for breast epithelial and not stromal cells. Specific cell-surface receptors for endothelin and biochemical responsiveness to the peptide were observed only in the stromal cells.

Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3.

The primary structure surrounding the residue on Inhibitor-2 phosphorylated by glycogen synthase kinase-3 has been determined. The sequence is: Lys-Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-His-Ser. This finding will facilitate studies of the effects of hormones on the phosphorylation state of Inhibitor-2 in vivo.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition.

Cadherin-13 (CDH13), a unique glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules, has been identified as a risk gene for attention-deficit/hyperactivity disorder (ADHD) and various comorbid neurodevelopmental and psychiatric conditions, including depression, substance abuse, autism spectrum disorder and violent behavior, while the mechanism whereby CDH13 dysfunction influences pathogenesis of neuropsychiatric disorders remains elusive. Here we explored the potential role of CDH13 in the inhibitory modulation of brain activity by investigating synaptic function of GABAergic interneurons. Cellular and subcellular distribution of CDH13 was analyzed in the murine hippocampus and a mouse model with a targeted inactivation of Cdh13 was generated to evaluate how CDH13 modulates synaptic activity of hippocampal interneurons and behavioral domains related to psychopathologic (endo)phenotypes. We show that CDH13 expression in the cornu ammonis (CA) region of the hippocampus is confined to distinct classes of interneurons. Specifically, CDH13 is expressed by numerous parvalbumin and somatostatin-expressing interneurons located in the stratum oriens, where it localizes to both the soma and the presynaptic compartment. Cdh13(-/-) mice show an increase in basal inhibitory, but not excitatory, synaptic transmission in CA1 pyramidal neurons. Associated with these alterations in hippocampal function, Cdh13(-/-) mice display deficits in learning and memory. Taken together, our results indicate that CDH13 is a negative regulator of inhibitory synapses in the hippocampus, and provide insights into how CDH13 dysfunction may contribute to the excitatory/inhibitory imbalance observed in neurodevelopmental disorders, such as ADHD and autism.

Calmodulin regulation of adenylate cyclase activity in human platelet membranes.

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.

Enhanced platelet cyclic AMP response to prostaglandin E1 in essential hypertension.

Platelets provide an accessible and homogeneous cellular system for investigative studies on hypertension. Hypertension-associated abnormalities of cyclic adenosine 3',5'-monophosphate (AMP) metabolism were studied in human platelets. Platelets from hypertensive subjects had an enhanced cyclic AMP accumulation response to prostaglandin E1 (twofold increase in prostaglandin E1 sensitivity). The degree of adenylate cyclase activation in response to both prostaglandin E1 (receptor-mediated) and forskolin (non-receptor-mediated) was greater in hypertensive than normotensive subjects, and prostaglandin E1-stimulated and forskolin-stimulated adenylate cyclase activity correlated directly (r = 0.71, p less than 0.001, n = 26). This finding suggests that the catalytic subunit of the enzyme is the rate-limiting step of this hormonal information transduction. Platelets from hypertensive subjects were more sensitive to epinephrine-induced inhibition of the stimulatory effects of prostaglandin E1 on both cyclic AMP accumulation (fourfold) and activation of cyclic AMP-dependent protein kinase. These findings suggest that the enhanced cyclic AMP metabolic response to prostaglandin E1 in platelets from subjects with established essential hypertension may function as a negative feedback mechanism to protect the cells against calcium overload and to reduce their stimulated participation in hemostatic and thrombotic processes.

Platelet membrane calmodulin-stimulated calcium-adenosine triphosphatase. Altered activity in essential hypertension.

Platelet free Ca2+ concentration has been found to be elevated in essential hypertension and to correlate with blood pressure level. Free cytoplasmic calcium concentration is determined by calcium influx, pooling, and efflux. The present study found a Ca2+-ATPase in platelet membranes that has a high affinity for Ca2+ (Km approximately 1 microM), is inhibited by low concentrations of orthovanadate (Ki approximately 1 microM), and can be stimulated by calmodulin (Km approximately 5 nM). The absolute increase in calmodulin-stimulated Ca2+-ATPase activity was not different between normotensive and hypertensive subjects; however, the degree of stimulation of Ca2+-ATPase activity at saturating calmodulin concentrations apparently was diminished in calmodulin-deficient membranes from subjects with established essential hypertension (40%) as compared to that in normotensive subjects of similar age (135%; p less than 0.001). Affinities for calmodulin and Ca2+ were comparable between the two groups, while the capacity for Ca2+-ATPase activity (basal and calmodulin-stimulated) was markedly greater (1.5- to 1.8-fold) in both native and calmodulin-deficient membranes from hypertensive subjects. On the other hand, the defective calcium efflux pump activity, as assessed by a decreased degree of calmodulin stimulation, may have contributed to elevated cytoplasmic calcium concentrations and the associated enhanced hormone sensitivity in platelets from essential hypertensive subjects. This may represent an adaptive negative feedback control mechanism to protect the cell against Ca2+ overload.

Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C.

This study compares vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats with respect to protein kinase C and intracellular responses to angiotensin II (Ang II). Ang II-induced degradation of polyphosphoinositides and accumulation of inositol di- and tris-phosphates was enhanced (approximately twofold) in hypertensive-derived cells, without a change (vs. normotensive-derived cells) in half-maximally effective concentrations of Ang II. Intracellular pH (approximately 6.6) was comparable between both cell isolates at quiescence, but alkalinization induced by Ang II, serum, or phorbol ester was greater (delta 0.1-0.2 pH units) for hypertensive-derived cells. For both cell types, the intracellular pH response to these agonists was prevented in the presence of Na+-H+ exchange inhibitors. S6 kinase activation induced by Ang II was enhanced (approximately twofold) in hypertensive-derived cells, whereas activation in response to serum or 12-O-tetradecanoylphorbol 13-acetate did not differ significantly between the two cell types. Quantitation of protein kinase C by immunoblotting and [3H]phorbol dibutyrate binding procedures revealed no differences between the two smooth muscle cell isolates (at quiescence or in the presence of serum) with respect to either total amounts or subcellular distribution. Sensitivity of protein kinase C to phorbol ester was apparently also not different between the two cell types, as assessed from dose-dependent (phorbol ester) S6 kinase activation profiles. Phorbol ester caused a similar subcellular redistribution of [3H]phorbol dibutyrate binding in the two cell isolates, but for both, minimal (10%) translocation occurred in response to Ang II. The data suggest that enhanced agonist responsiveness in vascular smooth muscle cells is unlikely to involve alterations in protein kinase C.

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

Aortic smooth muscle cells from spontaneously hypertensive rats (SHR) exhibit inappropriate proliferation characteristics in culture that suggest a modified response to serum mitogens or growth factors. The present study compares vascular smooth muscle cells from SHR and normotensive Wistar-Kyoto (WKY) rats with respect to their proliferative and functional response to growth factors. Specific attention was focused on the interaction of these vascular smooth muscle cells with epidermal growth factor. An increased growth rate of vascular smooth muscle cells from SHR (vs. WKY rats) was observed when cells were cultured in the presence of serum (10% and 0.5%), but not under serum-free conditions. The additional presence of low serum concentrations (0.5%) was required for epidermal growth factor to elicit a proliferative response, whereupon smooth muscle cells from SHR displayed an increased (vs. WKY rats) growth rate. Saturation binding of [125I]epidermal growth factor to intact smooth muscle cells indicated a twofold increase in receptor density in SHR-derived cells (p less than 0.001 vs. WKY rats) without an alteration in affinity for the growth factor. Cells derived from SHR also exhibited greater functional responsiveness to epidermal growth factor when compared with smooth muscle cells from WKY rats as evidenced by amplifications of both S6 kinase activation, phosphoinositide catabolism, elevation of intracellular pH, and DNA synthesis (nuclear labeling). We conclude that increased responsiveness of SHR-derived smooth muscle cells to epidermal growth factor could contribute to alterations in vascular smooth muscle growth and tone that may be fundamental to the pathogenesis of hypertension and atherosclerosis.

Vascular smooth muscle cell calcium fluxes. Regulation by angiotensin II and lipoproteins.

This study examined 45Ca uptake, 45Ca efflux, and the distribution of exchangeable 45Ca in confluent, quiescent cultures of aortic smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). These parameters were investigated under basal conditions and after addition of angiotensin II (Ang II) and low (LDL) and high (HDL) density lipoproteins. Basal 45Ca uptake was approximately 50% greater in VSMCs from SHRs (p < 0.005 versus WKY). Calcium antagonists (diltiazem or nifedipine) abolished this difference. The 45Ca uptake response to Ang II was approximately twofold greater in SHR than in WKY VSMCs (p < 0.05), and Ang II-induced increments of 45Ca uptake were weakly inhibited (by approximately 15-25%) by calcium antagonists. Lipoproteins also stimulated 45Ca uptake in VSMCs, and the apparent affinity of this process was approximately fivefold greater for LDL than for HDL. Calcium antagonists did not inhibit either LDL- or HDL-induced 45Ca uptake. SHR and WKY VSMCs did not differ with respect to 45Ca uptake induced by either LDL or HDL. The initial size of the slowly exchangeable pool of intracellular Ca2+ was approximately 35% greater in SHR VSMCs (p < 0.05 versus WKY). Ang II-induced mobilization of intracellular calcium (measured as the decrease in 45Ca content of the slowly exchangeable pool) was threefold greater in SHR VSMCs (p < 0.005 versus WKY). LDL and HDL marginally stimulated 45Ca efflux from this pool (< or = 20% above control) and to comparable extents in both SHR and WKY VSMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism.

A Mg-ATP-dependent protein phosphatase has been reconstituted from the catalytic subunit of protein phosphatase-1 and inhibitor-2, and consists of a 1:1 complex between these proteins. Activation of this enzyme by glycogen synthase kinase-3 and Mg-ATP results from the phosphorylation of inhibitor-2 on a threonine residue(s) and is accompanied by the dissociation of the complex. The results prove that protein phosphatase-1 and the Mg-ATP-dependent protein phosphatase contain the same catalytic subunit, and that they are interconvertible forms of the same enzyme.

Activation of human peripheral monocytes by angiotensin II.

This study has investigated the ability of the vasoconstrictor peptide angiotensin II to activate human peripheral blood monocytes. Activation was monitored by measuring both the release of tumor necrosis factor alpha from monocytes and their adhesion to monolayers of human endothelial cells. Angiotensin II-elicited activation of monocytes was dose-dependent (half-maximally effective concentration approximately 0.2 nM), saturable (maximally effective concentration approximately 5 nM), and sensitive to inhibition by the angiotensin type 1 receptor antagonist ZD 7155. Such direct actions imply that angiotensin II is an important candidate stimulus for the subendothelial infiltration of monocytes observed in atherogenesis and hypertension.

Density- and proliferation status-dependent expression of T-cadherin, a novel lipoprotein-binding glycoprotein: a function in negative regulation of smooth muscle cell growth?

The atypical low density lipoprotein (LDL) binding proteins (Mr 105 and 130 kDa; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T-cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T-cadherin and the density and proliferation status of SMC. T-cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand-binding techniques. T-cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T-cadherin levels were reversibly modulated by switching cultures between serum-free (upmodulation) and serum-containing (downmodulation) conditions. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF) or insulin-like growth factor (IGF) elicited a dose- and time-dependent downmodulation that was reversible after transfer of SMC to growth factor-free medium. Our results support the hypothesis that T-cadherin may function as a negative determinant of cell growth.

T-cadherin and signal-transducing molecules co-localize in caveolin-rich membrane domains of vascular smooth muscle cells.

Cadherins are a family of cellular adhesion proteins mediating homotypic cell-cell binding. In contrast to classical cadherins, T-cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T-cadherin might function as a signal-transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X-100 solubilization and density gradient centrifugation techniques. We demonstrate that T-cadherin is enriched in a minor detergent-insoluble low-density membrane domain and co-distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI-anchored proteins (CD-59, uPA receptor) and signal-transducing molecules (G alpha s protein and Src-family kinases), but completely excluded cell-cell and cell-matrix adhesion molecules (N-cadherin and beta1-integrin). Coupling of T-cadherin with signalling molecules within caveolae might enable cellular signal transduction.

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.

Identification of an atypical lipoprotein-binding protein from human aortic smooth muscle as T-cadherin.

We have previously described an atypical lipoprotein-binding protein of about 105 kDa (p105) in membranes of vascular smooth muscle cells (VSMCs) that is distinct from currently known lipoprotein receptors. In the present work we have developed a procedure for purification of p105 from human aortic media. Partial sequencing of purified protein has revealed identity of p105 with human T-cadherin. Anti-peptide antisera raised against human T-cadherin recognized a protein spot corresponding to the purified p105 on two-dimensional Western blots. The antisera also inhibited LDL binding to p105 on ligand blots. We conclude that the 105 kDa lipoprotein-binding protein present in human VSMCs is T-cadherin, an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell-cell adhesion proteins.

Effects of peptide vasoconstrictors on vessel structure.

The peptide vasoconstrictors angiotensin II and endothelin-1, originally described as being derived exclusively from the plasma renin-angiotensin system and vascular endothelium, respectively, have been demonstrated to be produced independently of these sources. Local tissue angiotensin-generating systems are well documented and endothelin production has been demonstrated for a variety of nonendothelial cells, including vascular smooth muscle cells. There is increasing evidence that these locally produced vasoconstrictor peptides may contribute to blood vessel homeostasis, as well as the development of vascular pathologic conditions. Results obtained from pharmaceutical intervention in humans and animals of these systems strongly support this hypothesis. In addition to their vasoconstrictor properties, angiotensin II and endothelin-1 act as potent biologic effectors. In vitro, both vasoconstrictor peptides appear to modulate the activity of autocrine feedback loops in vascular smooth muscle cells. The activity of these feedback loops in vivo may represent a central mechanism for regulation and phenotypic differentiation of this cell type. The most well-established autocrine feedback loops of vascular smooth muscle cells are constituted by platelet-derived growth factor and transforming growth factor-beta, both of which are influenced by the action of angiotensin II and endothelin-1. The effects of the peptide vasoconstrictors on the (auto-) regulated feedback loops are of long-term structural importance, since both vasoconstrictors (via autocrine growth modulators) may influence the composition of the extracellular matrix of vascular smooth muscle cells. This includes effects on the synthesis and secretion of thrombospondin, fibronectin, tenascin, etc. The secretion of extracellular matrix glycoproteins themselves and incorporation into extracellular matrix in vitro appear to be linked to the activity of the autocrine feedback loops: e.g., stimulation of thrombospondin mRNA results in secretion of the glycoprotein only in the concomitant presence of exogenous platelet-derived growth factor, whereas the expression of fibronectin and tenascin may be directed by transforming growth factor-beta. The influence of angiotensin II and endothelin-1 on vascular smooth muscle cell surface receptor expression may represent a secondary mode of action of these vasoconstrictor peptides. Endothelin-1, for instance, can rapidly down-regulate platelet-derived growth factor-alpha receptor mRNA and both angiotensin II and endothelin-1, via induction of transforming growth factor-beta, may interrupt the platelet-derived growth factor based autocrine feedback loop. In vivo, the highly complex interactions between local and systemic vasoconstrictor production, autoregulated feedback loops, and extracellular matrix (which also serves as a reservoir for growth and differentiation modulators) are central to vessel homeostasis.(ABSTRACT TRUNCATED AT 400 WORDS)

Na(+)-K+ pump and Na(+)-K+ co-transport in cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive rats: baseline activity and regulation.

OBJECTIVE: This paper examines the hypothesis that aberrations in vascular smooth muscle univalent ion transport systems play an important role in the pathogenesis of hypertension. DESIGN: Baseline Na(+)-K+ pump and Na(+)-K(+)-2Cl- co-transport activities and the regulation of these ion transport systems by angiotensin II and second messenger molecules have been studied in cultured aortic smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Ion transport was studied using isotopic univalent cations (86Rb and 22Na). RESULTS: Baseline Na(+)-K+ pump activity was comparable between SHR- and WKY-derived VSMC. Baseline Na(+)-K(+)-2Cl- and K(+)-Cl- co-transport activity as well as K+ leakage were significantly greater in SHR VSMC. Baseline Na(+)-K(+)-2Cl- co-transport was sensitive to inhibition by forskolin and ethyleneglycol-bis-(beta-amino ethylester)-N,N,N',N'-tetraacetic acid, whereas cyclic guanosine monophosphate and phorbol 12-myristate, 13-acetate had no effect. Angiotensin II-stimulated Na(+)-K(+)-2Cl- co-transport activity did not differ between WKY and SHR VSMC. Angiotensin II increased Na(+)-K(+)-pump activity to a significantly greater extent in SHR VSMC. The stimulatory effect of angiotensin II upon Na(+)-K+ pump activity was reduced under Na(+)-free buffer conditions and in the presence of the Na(+)-H+ exchange inhibitor, ethylisopropyl amiloride. Na(+)-K+ pump activity was also stimulated by the protein kinase C activator, phorbol 12-myristate, 13-acetate, and this was completely inhibited under Na(+)-free buffer conditions. CONCLUSIONS: SHR VSMC exhibit anomalous Na(+)-K(+)-pump and Na(+)-K(+)-2Cl- co-transport activities. The influence of these univalent ion transport systems upon cellular Na+ and Ca2+ homeostasis invoke their participation in the pathogenesis of hypertension.

Effect of Ox-LDL on endothelium-dependent response in pig ciliary artery: prevention by an ET(A) antagonist.

PURPOSE: To investigate whether oxidized low-density lipoprotein (Ox-LDL) affects endothelium-dependent responses in isolated porcine ciliary arteries. METHODS: In a myograph system for isometric force measurements, quiescent vessels were incubated with 50 microg/ml, 100 microg/ml, or 200 microg/ml Ox-LDL; 100 microg/ml native LDL (n-LDL); 1 microM of the ET(A)- endothelin receptor antagonist BQ 123; 100 microg/ml Ox-LDL coadministered with 1 microM BQ 123; or 100 microg/ml Ox-LDL coadministered with 50 microM of the protein synthesis inhibitor cycloheximide. Vessels with nonfunctional endothelium (intentionally and mechanically damaged) were also exposed to 100 microg/ml Ox-LDL. Two hours later, vessels were washed, precontracted with the thromboxane A2 analog U 46619 (approximately 0.1 microM), and exposed to bradykinin (0.1 nM to 3 microM), an endothelium-dependent relaxing agent. RESULTS: In quiescent vessels, Ox-LDL evoked delayed contractions. In contrast, no contractions were observed after exposure to n-LDL, BQ 123, Ox-LDL with BQ 123, or Ox-LDL with cycloheximide. In vessels with nonfunctional endothelium, Ox-LDL did not evoke contraction. Bradykinin-induced relaxations were inhibited in a dose-dependent manner by Ox-LDL, but not by n-LDL, BQ 123 alone, Ox-LDL with BQ 123, or Ox-LDL with cycloheximide. CONCLUSIONS: In porcine ciliary arteries, Ox-LDL affects endothelium-dependent responses through the activation of ET(A)- endothelin receptors. As Ox-LDL can accumulate in atherosclerotic plaques, such a mechanism might be involved in the occlusion of the ophthalmic circulation observed in patients with hypercholesterolemia and atherosclerosis.