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We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37[degrees]C) on an inverted fluorescence microscope. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum [Ca.sup.2+]-adenosine triphosphatase inhibitor, was used to deplete intracellular [Ca.sup.2+] stores after removing extracellular [Ca.sup.2+]. CCE was then activated by restoring extracellular [Ca.sup.2+] (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [[[Ca.sup.2+]].sub.i]. Restoring extracellular [Ca.sup.2+] caused a rapid peak increase in [[[Ca.sup.2+]].sub.i], followed by a sustained increase in [[[Ca.sup.2+]].sub.i]; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. protein kinase C; tyrosine kinase

Pulmonary veins (PV) make a significant contribution to total pulmonary vascular resistance. We investigated the cellular mechanisms by which the intravenous anesthetics propofol and thiopental alter adenosine triphosphate-sensitive potassium ([K.sup.+.sub.ATP]) channel relaxation in canine PV. The effects of [K.sup.+.sub.ATP] channel inhibition (glybenclamide), cyclooxygenase inhibition (indomethacin), nitric oxide synthase inhibition (L-NAME), and L-type voltage-gated [Ca.sup.2+] channel inhibition (nifedipine) on vasorelaxation responses to levcromakalim([K.sup.+.sub.ATP] channel activator) alone and in combination with the anesthetics were assessed. The maximal relaxation response to levcromakalim was attenuated by removing the endothelium and by L-NAME, but not by indomethacin. Propofol ([10.sup.-5], 3 x [10.sup.-5], and [10.sup.-4] M) and thiopental ([10.sup.-4] and 3 x [10.sup.-4] M) each attenuated levcromakalim relaxation in endothelium-intact (E+) rings, whereas propofol (3 x [10.sup.-5] and [10.sup.-4] M) and thiopental (3 x [10.sup.-4] M) attenuated levcromakalim relaxation in endothelium-denuded (E-) rings. In E+ rings, the anesthesia-induced attenuation of levcromakalim relaxation was decreased after pretreatment with L-NAME but not with indomethacin. In E-strips, propofol ([10.sup.-4] M) and thiopental (3 x [10.sup.-4] M) inhibited decreases in tension and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in response to levcromakalim, and these changes were abolished by nifedipine. These findings indicate that propofol and thiopental attenuate the endothelium-dependent component of [K.sup.+.sub.ATP] channel-induced PV vasorelaxation via an inhibitory effect on the nitric oxide pathway. Both anesthetics also attenuate the PV smooth muscle component of [K.sup.+.sub.ATP] channel-induced relaxation by reducing the levcromakalim-induced decrease in [[[Ca.sup.2+]].sub.i] via an inhibitory effect on L-type voltage-gated [Ca.sup.2+] channels. [Ca.sup.2+] influx; anesthetics

Our objectives were to identify the relative contributions of [[[Ca.sup.2+]].sub.i] and myofilament [Ca.sup.2+] sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane [A.sub.2] mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-46619-induced contraction in PVSM is mediated by both increases in [[[Ca.sup.2+]].sub.i] and myofilament [Ca.sup.2+] sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) tings. In addition, [[[Ca.sup.2+]].sub.i] and tension were simultaneously measured in fura-2-1oaded E- PVSM strips. U-46619 (0.1 nM-1 [micro]M) caused dose-dependent (P < 0.001) contraction in PV tings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated [Ca.sup.2+] channels (nifedipine, P < 0.001), inositol 1,4,5-trisphosphate-mediated [Ca.sup.2+] release (2-aminoethoxydiphenylborate, P < 0.001), PKC (bisindolylmaleimide I, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [[[Ca.sup.2+]].sub.i] and myofilament [Ca.sup.2+] sensitivity. Both [Ca.sup.2+] influx and release mediated the early transient increase in [[[Ca.sup.2+]].sub.i], whereas the late sustained increase in [[[Ca.sup.2+]].sub.i] only involved [Ca.sup.2+] influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-46619-induced increase in myofilament [Ca.sup.2+] sensitivity. These results suggest that U-46619 contraction is mediated by [Ca.sup.2+] influx, [Ca.sup.2+] release, and increased myofilament [Ca.sup.2+] sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction. vein; [Ca.sup.2+] homeostasis; myofilament [Ca.sup.2+] sensitivity

We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) were measured simultaneously in fura-2-loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 [micro]M) caused dose-dependent contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated [Ca.sup.2+] channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (I[P.sub.3])-mediated [Ca.sup.2+] release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolyl-maleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E- PV rings. ACh (1 [micro]M) caused a leftward shift in the [[[Ca.sup.2+]].sub.i]-tension relationship (P = 0.015), i.e., ACh increased myofilament [Ca.sup.2+] sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament [Ca.sup.2+] sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves [Ca.sup.2+] influx through voltage-operated [Ca.sup.2+] channels and I[P.sub.3]-mediated [Ca.sup.2+] release. In addition, ACh induces increased myofilament [Ca.sup.2+] sensitivity, which requires the PKC, Rho-kinase, and TK pathways. endothelium; nitric oxide; myofilament calcium sensitivity; calcium influx and release

Our objectives were to identify the relative contributions of intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and myofilament [Ca.sup.2+] sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the [alpha]-adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [[[Ca.sup.2+]].sub.i], myofilament [Ca.sup.2+] sensitization, and vasomotor tone in response to [alpha]-adrenoreceptor stimulation of PASM. Simultaneous measurement of [[[Ca.sup.2+]].sub.i] and isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal [Ca.sup.2+] influx, [Ca.sup.2+] release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum [Ca.sup.2+] stores, and myofilament [Ca.sup.2+] sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal [Ca.sup.2+] influx component and the myofilament [Ca.sup.2+] sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced [Ca.sup.2+] sensitization. ROK inhibition slightly decreased [[[Ca.sup.2+]].sub.i] but completely inhibited myofilament [Ca.sup.2+] sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal [Ca.sup.2+] influx in response to [alpha]-adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament [Ca.sup.2+] sensitivity. ROK is the predominant pathway mediating PE-induced myofilament [Ca.sup.2+] sensitization. intracellular calcium ion; myofilament calcium ion sensitivity; phenylephrine; vascular smooth muscle; pulmonary artery smooth muscle; protein kinase C

A study was conducted to analyze the effects of left lung autotransplantation (LLA) on the pulmonary vasorelaxant response to the sympathetic beta-adrenoreceptor agonist isoproterenol and to determine the cellular mechanism involved in any of the resulting effect. To examine the specific effects of surgical transplantation on mechanisms of pulmonary vascular regulation, an experimental model of LLA was employed. The results are discussed.

The cellular mechanism that contribute to the attenuation in nitric oxide (NO)-mediated pulmonary vasorelaxation post-left lung autotransplantation (LLA) is analyzed. The analysis was undertaken by determining the pulmonary vasorelaxant responses to the endothelial agonists ACh and calcium ionophore. The findings show that the attenuated pulmonary vasodilation post-LLA engages inactivation of NO by superoxide anion caused by endothelial xanthine oxidase.

The beat to beat variation in the R-R interval of the electrocardiogram of conscious dogs was investigated under different conditions using sympathetic and parasympathetic blocking agents alone or in combination to determine the entropy measures of heart rate variability. Results reveal the the entropy of the variability in the heart rate of conscious dogs is brought about by the parasympathetic control of heart rate.

Protein kinase C (PKC) isoform-specific antibodies, Western blot analyses, indirect immunofluorescence and confocal microscopy were used to determine the pattern of PKC activation and distribution in cultured pulmonary artery smooth muscle cells from dogs. Results reveal that the interaction of angiotensin II on cellular receptors triggers a cascade that results in the upregulation of specific PKC isoforms and the translocation of these isoforms to particular areas in the cell.

Chronically instrumented mongrel dogs were analyzed after the administration of isoflurane anesthesia to determine the effects of the anesthetic on the activation of endothelium-dependent pulmonary vasodilation. Administration of isoflurane in chronically instrumented mongrel dogs selectively inhibited endothelium-dependent vasodilator sensitivity to bradykinin. However, administration of halothane did not alter vasodilation induced by 3-morpholinosydonimine-N-ethylcarbamide.

Study of endothelial and vascular smooth muscle responses suggests that left lung autotransplantation (LLA) increases sensitivity of vascular smooth muscle to alpha1-adrenergic activation and endothelial dysfunction. LLA reduces the activity of endothelium-derived relaxing factor/nitric oxide which improves vascular resistance in transplanted lungs. Relaxation to SIN-1, an NO donor, is identical for arterial rings and LLA lungs.

A study was conducted to examine to what extent the endothelium dependent and endothelium independent vasodilation in the pulmonary circulation of conscious dogs is changed by left lung autotransplantation (LLA). Considerable damage in vasolidation which is endothelium dependent is connected with LLA, but vasodilation which is not dependent on endothelium is unchanged compared with controlled conscious dogs. An important part of the continuous movement in the pulmonary vascular pressure-flow (LPQ) relationship post-LLA is passive and does not show an enhancement in baseline vasomotor tone.

Doi, Shouzaburoh, Derek S. Damron, Koji Ogawa, Satoru Tanaka, Mayumi Horibe, and Paul A. Murray. [K.sup.+] channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle. Am J Physiol Lung Cell Mol Physiol 279: L242-L251, 2000.--We investigated the role of [K.sup.+] channels in the regulation of baseline intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), [Alpha]-adrenoreceptor-mediated [Ca.sup.2+] signaling, and capacitative [Ca.sup.2+] entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated [K.sup.+] channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [[[Ca.sup.2+]].sub.i] but attenuated the amplitude and frequency of the [[[Ca.sup.2+]].sub.i] oscillations induced by the [Alpha]-agonist phenylephrine (PE). Inhibition of [Ca.sup.2+]-activated [K.sup.+] channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive [K.sup.+] channels (with lemakalim) had no effect on resting [[[Ca.sup.2+]].sub.i] or PE-induced [[[Ca.sup.2+]].sub.i] oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum [Ca.sup.2+] stores in the absence of extracellular [Ca.sup.2+]. Under these conditions, 4-AP attenuated the peak and sustained components of capacitative [Ca.sup.2+] entry, which was observed when extracellular [Ca.sup.2+] was restored. Capacitative [Ca.sup.2+] entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated [K.sup.+] channel inhibition increases resting [[[Ca.sup.2+]].sub.i] and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative [Ca.sup.2+] entry. [Alpha]-adrenoreceptor activation; intracellular calcium; voltage-gated potassium channels; pulmonary circulation; vasoconstriction

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37°C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration ([Ca22+]i) was measured as the 340/380 fluorescence ratio in individ- ual PASMCs. Thapsigargin, a sarcoplasmic reticulum Ca2+adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+ CCE was then activated by restoring extracellular Ca2+ (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [Ca2+]i. Restoring extracellular Ca22+ caused a rapid peak increase in [Ca22+]1, followed by a sustained increase in [Ca22+],; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. [ABSTRACT FROM AUTHOR]

Pulmonary veins (PV) make a significant contribution to total pulmonary vascular resistance. We investigated the cellular mechanisms by which the intravenous anesthetics propofol and thiopental alter adenosine triphosphate-sensitive potassium (KATP+) channel relaxation in canine PV. The effects of KATP+ channel inhibition (glybenclamide), cyclooxygenase inhibition (indomethacin), nitric oxide synthase inhibition (L-NAME), and L-type voltage-gated Ca2+ channel inhibition (nifedipine) on vasorelaxation responses to levcromakalim (KATP+ channel activator) alone and in combination with the anesthetics were assessed. The maximal relaxation response to levcromakalim was attenuated by removing the endothelium and by L-NAME, but not by indomethacin. Propofol (10-5, 3 × 10-5, and 10-4 M) and thiopental (10-4 and 3 × 10-4 M) each attenuated levcromakalim relaxation in endothelium-intact (E+) rings, whereas propofol (3 × 10-5 and 10-4 M) and thiopental (3 × 10-4 M) attenuated levcromakalim relaxation in endothelium-denuded (E-) rings. In E+ rings, the anesthesia-induced attenuation of levcromakalim relaxation was decreased after pretreatment with L-NAME but not with indomethacin. In E-strips, propofol (10-4 M) and thiopental (3 × 10-4 M) inhibited decreases in tension and intracellular Ca2+ concentration ([Ca2+]i) in response to levcromakalim, and these changes were abolished by nifedipine. These findings indicate that propofol and thiopental attenuate the endothelium-dependent component of KATP+ channel-induced PV vasorelaxation via an inhibitory effect on the nitric oxide pathway. Both anesthetics also attenuate the PV smooth muscle component of KATP+ channel-induced relaxation by reducing the levcromakalim-induced decrease in [Ca2+]i via an inhibitory effect on L-type voltage-gated Ca2+ channels. [ABSTRACT FROM AUTHOR]

Our objectives were to identify the relative contributions of [Ca2+]i and myofilament Ca2+ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A2 mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-466 19-induced contraction in PVSM is mediated by both increases in [Ca2+]i and myofilament Ca2+ sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) rings. In addition, [Ca2+]i and tension were simultaneously measured in fura-2-loaded E- PVSM strips. U-46619 (0.1 nM-1 ·used dose-dependent (P < 0.001) contraction in PV rings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol 1 ,4,5 -trisphosphate-mediated Ca2+ release (2-aminoethoxydiphenylborate, P< 0.001), PKC (bisindolylmaleimide 1, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. Both Ca2+ influx and release mediated the early transient increase in [Ca2+]i, whereas the late sustained increase in [Ca2+]i only involved Ca2+ influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-466 19-induced increase in myofilament Ca2+ sensitivity. These results suggest that U-46619 contraction is mediated by Ca2+ influx, Ca2+ release, and increased myofilament Ca2+ sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction. [ABSTRACT FROM AUTHOR]