Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways.

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both ß1- and ß2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by ß2-adrenoceptor stimulation than ß1-adrenoceptor stimulation. Both ß-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the ß-adrenoceptor-potentiated firing rate. The stimulatory effects of ß- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under ß-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Molecular identification of HSPA8 as an accessory protein of a hyperpolarization-activated chloride channel from rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are the major origin of atrial fibrillation. Recently, we recorded hyperpolarization-activated Cl- current (ICl, h) in rat PV cardiomyocytes. Unlike the well-known chloride channel protein 2 (CLCN2) current, the activation curve of ICl, h was hyperpolarized as the Cl- ion concentration ([Cl-] i ) increased. This current could account for spontaneous activity in PV cardiomyocytes linked to atrial fibrillation. In this study, we aimed to identify the channel underlying ICl, h Using RT-PCR amplification specific for Clcn2 or its homologs, a chloride channel was cloned from rat PV and detected in rat PV cardiomyocytes using immunocytochemistry. The gene sequence and electrophysiological functions of the protein were identical to those previously reported for Clcn2, with protein activity observed as a hyperpolarization-activated current by the patch-clamp method. However, the [Cl-] i dependence of activation was entirely different from the observed ICl, h of PV cardiomyocytes; the activation curve of the Clcn2-transfected cells shifted toward positive potential with increased [Cl-] i , whereas the ICl, h of PV and left ventricular cardiomyocytes showed a leftward shift. Therefore, we used MS to explore the possibility of additional proteins interacting with CLCN2 and identified an individual 71-kDa protein, HSPA8, that was strongly expressed in rat PV cardiomyocytes. With co-expression of HSPA8 in HEK293 and PC12 cells, the CLCN2 current showed voltage-dependent activation and shifted to negative potential with increasing [Cl-] i Molecular docking simulations further support an interaction between CLCN2 and HSPA8. These findings suggest that CLCN2 in rat heart contains HSPA8 as a unique accessory protein.

Purinergic receptor stimulation induces calcium oscillations and smooth muscle contraction in small pulmonary veins.

KEY POINTS: We investigated the excitation-contraction coupling mechanisms in small pulmonary veins (SPVs) in rat precision-cut lung slices. We found that SPVs contract strongly and reversibly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced vasoconstriction in SPVs was associated with the stimulation of purinergic P2Y2 receptors in vascular smooth muscle cell, activation of phospholipase C-ß and the generation of intracellular Ca2+ oscillations mediated by cyclic Ca2+ release events via the inositol 1,4,5-trisphosphate receptor. Active constriction of SPVs may play an important role in the development of pulmonary hypertension and pulmonary oedema. ABSTRACT: The small pulmonary veins (SPVs) may play a role in the development of pulmonary hypertension and pulmonary oedema via active changes in SPV diameter, mediated by vascular smooth muscle cell (VSMC) contraction. However, the excitation-contraction coupling mechanisms during vasoconstrictor stimulation remain poorly understood in these veins. We used rat precision-cut lung slices and phase-contrast and confocal microscopy to investigate dynamic changes in SPV cross-sectional luminal area and intracellular Ca2+ signalling in their VSMCs. We found that the SPV (∼150 µm in diameter) contract strongly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced SPV contraction was fast, concentration-dependent, completely reversible upon ATP washout, and inhibited by purinergic receptor antagonists suramin and AR-C118925 but not by MRS2179. Immunofluorescence showed purinergic P2Y2 receptors expressed in SPV VSMCs. ATP-induced SPV contraction was inhibited by phospholipase Cß inhibitor U73122 and accompanied by intracellular Ca2+ oscillations in the VSMCs. These Ca2+ oscillations and SPV contraction were inhibited by the inositol 1,4,5-trisphosphate receptor inhibitor 2-APB but not by ryanodine. The results of the present study suggest that ATP-induced vasoconstriction in SPVs is associated with the activation of purinergic P2Y2 receptors in VSMCs and the generation of Ca2+ oscillations.

Expression of vascular endothelial growth factor (VEGF)-B and its receptor (VEGFR1) in murine heart, lung and kidney.

Metabolic diseases, such as obesity and diabetes, are a serious burden for the health system. Vascular endothelial growth factor (VEGF)-B has been shown to regulate tissue uptake and accumulation of fatty acids and is thus involved in these metabolic diseases. However, the cell-type-specific expression pattern of Vegfb and its receptor (VEGFR1, gene Flt1) remains unclear. We explore the expression of Vegfb and Flt1 in the murine heart, lung and kidney by utilizing ß-galactosidase knock-in mouse models and combining the analysis of reporter gene expression and immunofluorescence microscopy. Furthermore, Flt1 heterozygous mice were analyzed with regard to muscular fatty acid accumulation and peripheral insulin sensitivity. Throughout the heart, Vegfb expression was found in cardiomyocytes with a postnatal ventricular shift corresponding to known changes in energy requirements. Vegfb expression was also found in the pulmonary myocardium of the lung and in renal epithelial cells of the thick ascending limb of Henle's loop, the connecting tubule and the collecting duct. In all analyzed organs, VEGFR1 expression was restricted to endothelial cells. We also show that reduced expression of VEGFR1 resulted in decreased cardiac fatty acid accumulation and increased peripheral insulin sensitivity, possibly as a result of attenuated VEGF-B/VEGFR1 signaling. Our data therefore support a tightly controlled, paracrine signaling mechanism of VEGF-B to VEGFR1. The identified cell-specific expression pattern of Vegfb and Flt1 might form the basis for the development of cell-type-targeted research models and contributes to the understanding of the physiological and pathological role of VEGF-B/VEGFR1 signaling.

Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms.

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

Optimized RNA extraction from non-deparaffinized, laser-microdissected material.

mRNA extraction and subsequent RT-polymerase chain reaction (PCR)-based expression analysis from laser-microdissected material is by now a well-established and reproducible method. Most routinely stored tissue samples are preserved as formalin-fixed, paraffin-embedded materials. While this allows for a convenient storage and stable preservation of nucleic acids, deparaffinization before staining for laser microdissection may result in a significant loss of mRNA quality and consequently of PCR sensitivity. We describe a method of isolating anatomic compartments from non-deparaffinized, formalin-fixed, and paraffin-embedded tissues by laser-assisted microdissection which allows for a highly efficient mRNA retrieval.

Phenylephrine contracts porcine pulmonary veins via alpha(1B)-, alpha(1D)-, and alpha(2)-adrenoceptors.

We have recently shown that the postjunctional alpha(2)-adrenoceptor mediating contraction of porcine pulmonary veins is of the alpha(2C)-subtype. We could also demonstrate that alpha(1)-adrenoceptors might contribute to the contraction in that blood vessel. In the present study, we aimed at characterising the alpha(1)-adrenoceptor subtype(s) involved using pharmacological and molecular biological methods. In isolated rings of porcine pulmonary veins the typical alpha(1)-adrenoceptor agonist phenylephrine caused a concentration-dependent contraction that was inhibited by the alpha(1B)-adrenoceptor selective antagonists 1-[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-2-[2-(isopropyl)-6-methoxyphenoxy]ethan-1-one (Rec15/2615; pA(2) 8.96+/-0.13) and 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pA(2) 7.22+/-0.05), as well as the alpha(1D)-adrenoceptor selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378; pA(2) 8.29+/-0.15, slope of the Schild plot 0.75+/-0.09, significantly different from unity, P<0.05), but not by the alpha(1A)-adrenoceptor selective antagonists (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine (RS-17053). These findings suggest that phenylephrine activates both alpha(1B)- and alpha(1D)-adrenoceptors. The observation was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) in porcine pulmonary veins, where mRNA signals for alpha(1B)- and alpha(1D)-adrenoceptors could be detected. However, the antagonist properties of rauwolscine and yohimbine (non-subtype selective alpha(2)-adrenoceptor antagonists) against phenylephrine showed that this agonist also activates alpha(2)-adrenoceptors in pulmonary veins. This was strengthened in experiments using tissues that were stimulated with forskolin (cell permeable activator of adenylyl cyclase). Phenylephrine mimicked the effect of the selective alpha(2)-adrenoceptor agonist UK14304 by causing an inhibition of forskolin-stimulated cAMP accumulation that was blocked by rauwolscine. It is concluded that, in addition to alpha(1B)- and alpha(1D)-adrenoceptors, phenylephrine can stimulate alpha(2)-adrenoceptors in porcine pulmonary veins.

Emerging pulmonary vasculature lacks fate specification.

Lung morphogenesis requires precise coordination between branching morphogenesis and vascularization to generate distal airways capable of supporting respiration at the cell-cell interface. The specific origins and types of blood vessels that initially form in the lung, however, remain obscure. Herein, we definitively show that during the early phases of lung development [i.e., embryonic day (E) 11.5], functional vessels, replete with blood flow, are restricted to the mesenchyme, distal to the epithelium. However, by day E14.5, and in response to epithelial-derived VEGF signals, functional vessels extend from the mesenchyme to the epithelial interface. Moreover, these vessels reside adjacent to multipotent mesenchymal stromal cells that likely play a regulatory role in this process. As well as and distinct from the systemic vasculature, immunostaining for EphrinB2 and EphB4 revealed that arterial and venous identity is not distinguishable in emergent pulmonary vasculature. Collectively, this study provides evidence that lung vascularization initially originates in the mesenchyme, distal to the epithelium, and that arterial-venous specification does not exist in the early lung. At a mechanistic level, we show that basilar epithelial VEGF prompts endothelial cells to move toward the epithelium where they undergo morphogenesis during the proliferative, canalicular stage. Thus our findings challenge existing notions of vascular origin and identity during development.

Interstitial Cajal-like cells (ICLC) in myocardial sleeves of human pulmonary veins.

We present here evidence for the existence of a new type of interstitial cell in human myocardial sleeves of pulmonary veins: interstitial Cajal-like cell (ICLC). This cell fulfils the criteria for positive diagnosis of ICLC, including CD 117/c-kit positivity. Transmission electron microscopy revealed typical ICLC with 2 or 3 very long processes (several tens of mm) suddenly emerging from the cellular body. Also, these processes appear moniliform but extremely thin (0.1-0.4 mm) under the resolving power of the usual microscopy. Cell processes establish close spatial relationships between each other, as well as with capillaries and nerve endings. ICLC appear located among the myocardial cells and particularly at the border between the myocardial sleeve and pulmonary vein wall.

Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.

Calcium-mediated triggered activity is an underlying cellular mechanism of ectopy originating from the pulmonary vein in dogs.

Paroxysmal atrial fibrillation associated with focal ectopy originating from the pulmonary vein (PV) can be preceded by variations in autonomic tone; however, the underlying cellular mechanisms are not clear. To determine the mechanisms of autonomically mediated PV ectopy, high-resolution optical mapping techniques were used to measure action potentials and Ca(2+) transients from the PV and the ligament of Marshall area in the arterially perfused canine left atrium. Rapid pacing was used to initiate ectopic activity during pituitary adenylate cyclase-activating polypeptide (PACAP) injection (1 nmol), as a surrogate for autonomic imbalance, before (n = 9) and after (n = 6) verapamil (10 nmol) administration. In all preparations, spontaneous activity was absent before rapid pacing. During PACAP injection, rapid pacing induced ectopic activity in eight of nine preparations. In contrast, before PACAP injection, rapid pacing did not induce ectopic activity. Activation maps of each episode of ectopic activity indicated that the site of origin occurred more frequently in the PV (70%) than in the ligament of Marshall (30%) area. As rapid pacing cycle length increased, so did the ectopic beat coupling interval. In addition, PACAP-induced ectopic activity was associated with large Ca(2+) transient amplitudes and was always suppressed by verapamil, a Ca(2+) channel blocker (P < 0.05). Finally, during PACAP injection in the absence of an ectopic beat, spontaneous Ca(2+) release and delayed afterdepolarizations were observed simultaneously after termination of rapid pacing. In conclusion, these data suggest that autonomically mediated PV ectopy may be due to Ca(2+)-mediated triggered activity arising from delayed afterdepolarizations.

Comparison of ion channel distribution and expression in cardiomyocytes of canine pulmonary veins versus left atrium.

BACKGROUND: Cardiomyocytes in pulmonary vein (PV) sleeves are important in atrial fibrillation (AF), but underlying mechanisms are poorly understood. Pulmonary veins have different ionic current properties compared to left atrium, with pulmonary vein inward-rectifier currents being smaller and delayed-rectifier currents larger than in left atrium. METHODS: Expression and distribution of the inward-rectifier subunits Kir2.1 and Kir2.3, the rapid delayed-rectifier alpha-subunit ERG, the slow delayed-rectifier alpha-subunit KvLQT1, the beta-subunit minK, the L-type Ca(2+)-subunit Ca(v)1.2, and the Na(+),Ca(2+)-exchanger were quantified by Western blot on isolated cardiomyocytes and localized by immunohistochemistry in tissue sections obtained from canine hearts. RESULTS: Western blotting indicated significantly greater expression of ERG (by 28%, P<0.05) and KvLQT1 (by 34%, P<0.05) in pulmonary vein versus left atrial (LA) cardiomyocytes, but smaller Kir2.3 and similar Kir2.1, Ca(v)1.2 and Na(+),Ca(2+)-exchanger expression in PV. Kir2.1 exhibited weak transverse tubular distribution in both regions. Kir2.3 localized to intercalated disks in both regions, and to transverse tubules in left atrium but not pulmonary vein. ERG staining was more intense in pulmonary vein than left atrium, localizing to transverse tubules in both regions and intercalated disks in pulmonary veins. KvLQT1 was more intensely expressed in pulmonary veins, with a transverse tubular and intercalated disk localization, versus a more diffuse signal in left atrium. The Na(+),Ca(2+)-exchanger localized to transverse tubules, plasma membranes and intercalated disks with similar intensity in each region. CONCLUSIONS: Greater ERG and KvLQT1 abundance in pulmonary vein cardiomyocytes, lower abundance of Kir2.3 in pulmonary veins and differential pulmonary vein subcellular distribution of Kir2.3, ERG and KvLQT1 subunits may contribute to ionic current differences between pulmonary vein and left atrial cardiomyocytes.

Potassium channels regulate tone in rat pulmonary veins.

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.

SQ22536 and W-7 inhibit forskolin-induced cAMP elevation but not relaxation in newborn ovine pulmonary veins.

The role of cAMP in forskolin-induced relaxation was studied in isolated pulmonary veins of newborn lambs (7-12 days). In vessels preconstricted with endothelin-1, forskolin at concentrations < or =10(-7) M had no effect on cAMP content and adenylyl cyclase activity but caused up to 50% relaxation. At higher concentrations, forskolin markedly elevated cAMP content and adenylyl cyclase activity and caused a further relaxation. SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor] and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide; a calmodulin-dependent adenylyl cyclase inhibitor] had no significant effect on forskolin-induced relaxation but markedly inhibited the elevation of cAMP content and adenylyl cyclase activity caused by forskolin. Rp-8-CPT-cAMPS [8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate; an inhibitor of cAMP-dependent protein kinases] and Rp-8-Br-PET-cGMPS (beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate; an inhibitor of cGMP-dependent protein kinases) attenuated the relaxation caused by a cAMP analog but not that caused by forskolin. These results suggest that cAMP may not play a major role in forskolin-induced relaxation of pulmonary veins of newborn lambs.

Cardiac and smooth muscle cell contribution to the formation of the murine pulmonary veins.

Previous studies have demonstrated that the primordial pulmonary veins originate as an outgrowth of the atrial cells and anastomosis with the pulmonary venous plexus. As a consequence of this embryologic origin the tunica media of these vessels is composed of cardiac cells that express atrial specific markers (Lyons et al. [1990] J Cell Biol 111:2427-2436; Jones et al. [1994] Dev Dyn 200:117-128). We used transgenic mice for the cardiac troponin I (cTNI) gene and smooth muscle (SM) myosin heavy chain as differentiation markers, to analyze how cardiac and SM cells contribute to the formation and structural remodeling of the pulmonary veins during development. We show here that the tunica media of the adult mouse pulmonary veins contains an outer layer of cardiac cells and an intermediate SM cell compartment lining down on the inner endothelium. This structural organization is well expressed in the intrapulmonary veins from the beginning of vasculogenesis, with cardiac cells accumulating over preexisting roots of endothelial and SM cells and extending to the third bifurcation of the pulmonary branches without reaching the more distal tips of the vessels. On the other hand, SM cells, which are widely distributed in the intrapulmonary veins from the embryonic stage E16, accumulate also in the extrapulmonary branches and reach the posterior wall of the left atrium, including the orifices of the pulmonary veins. This event takes place around birth when the pulmonary blood flow starts to function properly. A model for the development of the pulmonary veins is presented, based upon our analysis.

Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2.

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.

Distribution of atrial natriuretic polypeptide (ANP)-containing cells in the rat heart and pulmonary vein. Immunohistochemical study and radioimmunoassay.

The distribution of atrial natriuretic polypeptide (ANP) was immunohistochemically surveyed in the rat heart and lung using an antiserum raised against alpha-human ANP. The ANP-immunoreactive cells were seen to be distributed in the atrial walls and proximal portions of the pulmonary vein and venae cavae, but were absent from the aorta, pulmonary arteries, trachea, bronchus, and alveolar cells. The immunoreactive cells were present in a narrow region just beneath the endothelium of the pulmonary vein and vena cavae, and, ultrastructurally and immunocytochemically, were seen to be striated muscle cells with ANP-containing specific granules similar to those seen in atrial cardiocytes. A radioimmunoassay for ANP revealed a content of 604 +/- 51 pg/mg wet weight in the pulmonary vein, and 3343 +/- 1620 pg/mg wet weight in the venae cavae. In addition to the atrial wall, the proximal portion of both the pulmonary vein and venae cavae are suggested to be constituents of an ANP-producing organ.