Intracellular and extracellular calcium utilization during hypoxic vasoconstriction of cyclostome aortas.

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary and cyclostome aortic vascular smooth muscle. The present study examined the utilization of calcium during HV in dorsal aortas (DA) from sea lamprey and New Zealand hagfish. HV was temporally correlated with increased free cytosolic calcium (Ca2+c) in lamprey DA. Extracellular calcium (Ca2+o) did not contribute significantly to HV in lamprey DA, but it accounted for 38.1 +/- 5.3% of HV in hagfish DA. Treatment of lamprey DA with ionomycin, ryanodine, or caffeine added to thapsigargin-reduced HV, whereas HV was augmented by BAY K 8644. Methoxyverapamil (D600) in zero Ca2+o did not affect HV in lamprey DA, nor did it prevent further constriction when Ca2+o was restored during hypoxia in hagfish DA. Removal of extracellular sodium (Na+o) caused a constriction in both species. Lamprey DA relaxed to prehypoxic tension following return to normoxia in zero Na+o, whereas relaxation was inhibited in hagfish DA. Relaxation following HV was inhibited in lamprey DA when Na+o and Ca2+o were removed. These results show that HV is correlated with [Ca2+]c in lamprey DA and that Na+/Ca2+ exchange is used during HV in hagfish but not lamprey DA. Multiple receptor types appear to mediate stored intracellular calcium release in lamprey DA, and L-type calcium channels do not contribute significantly to constriction in either cyclostome.

Estrogen protects against hypertension in the spontaneously hypertensive rat, but its protective mechanism is unrelated to impaired arterial muscle relaxation.

OBJECTIVES: To determine if estrogen acutely and directly alters arterial muscle relaxation, if estrogen is responsible for gender dichotomy in hypertension, and if arterial muscle from female spontaneously hypertensive rats (SHR) is slow to relax as is muscle from male SHR compared with arterial muscle of normotensive Wistar-Kyoto rats (WKY). METHODS: Relaxation rates of isometrically contracted arterial muscle from male rats were measured before and after addition of beta-estradiol. Blood pressure (BP) was monitored in intact male and female SHR and WKY and in ovariectomized and estrone-treated ovariectomized SHR and WKY. Relaxation rates of maximum isometric contractions of arterial muscle excised from male SHR and WKY and female SHR and WKY with varying chronic estrogen status were measured. RESULTS: Beta-estradiol had no direct, acute effect on arterial muscle force or relaxation. Intact and estrone-implanted SHR females had significantly lower BP than males. Ovariectomized SHR developed high BP equivalent to that of males. Arterial relaxation was slower in both male and female SHR compared with WKY. CONCLUSIONS: Estrogen lowers BP in female SHR. Strain differences in relaxation rates are independent of gender and estrogen status. Estrogen has no effect on arterial muscle relaxation, suggesting another mechanism for the protective effect of estrogen in hypertension.

H(2)O(2) mediates Ca(2+)- and MLC(20) phosphorylation-independent contraction in intact and permeabilized vascular muscle.

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H(2)O(2). Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H(2)O(2). A second purpose was to determine whether H(2)O(2)-induced contraction is calcium independent. The addition of H(2)O(2) to calcium-depleted (using the Ca(2+) ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H(2)O(2) even in zero Ca(2+). The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC(20)) phosphorylation plays a role in H(2)O(2)-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H(2)O(2)-induced contractions, and the relative amounts of phosphorylated MLC(20) were measured. H(2)O(2) caused dose-dependent contractions that were independent of MLC(20) phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H(2)O(2) contractile response. In conclusion, H(2)O(2) induces Ca(2+)- and MLC(20) phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction.

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.

Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction.

The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smoothmuscle (BCASM) stimulated with phorbol 12,13-dibutyrate (PDBu) under Ca2+-containing and Ca2+-free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca2+. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca2+-free conditions. Immunoblot analysis of BCASM using PKC isoform-specific antibodies demonstrated the presence of one "Ca2+- dependent" PKC isoform: alpha, and two of the "Ca2+-independent" isoforms: epsilon and zeta. These data suggest that Ca2+-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation.

Myosin isoform shifts and decreased reactivity in hypoxia-induced hypertensive pulmonary arterial muscle.

The principal stimulus that evokes pulmonary hypertension is chronic alveolar hypoxia. Pulmonary hypertension is associated with remodeling of the vessel walls, involving hypertrophy and hyperplasia of pulmonary arterial smooth muscle (PASM) and a concomitant increase in the deposition of connective tissue, resulting in increased wall thickness. The purpose of the present study was to determine the effect of hypoxia-induced hypertension on the structure and function of PASM. Experiments were designed to determine whether hypoxia-induced pulmonary hypertension is associated with alterations in PASM: 1) reactivity to a variety of agonists, 2) contractile protein proportions and isoforms, and 3) structural properties. Young adult male rats were made hypoxic by lowering the fraction of inspired O2 (10%) for 14 days. Pulmonary arterial segments were isolated and dose-response curves to various agonists (high K+, norepinephrine, serotonin, angiotensin II, and adenosine) were generated. Gel electrophoresis was used to measure changes in the relative amounts of actin or myosin and of myosin heavy chain (MHC) isoforms. Structural changes were correlated with the pharmacological and biochemical data. Hypoxia-induced pulmonary hypertension caused a general decreased reactivity, an increase in the proportion of nonmuscle to muscle MHC isoforms in PASM, and an increase in arterial wall thickness with PASM hypertrophy or hyperplasia.

Arterial muscle myosin heavy chains and light chains in spontaneous hypertension.

Increased maximum velocity of shortening (Vmax), increased shortening ability (delta Lmax) and decreased relaxation rate have been reported for arterial smooth muscle from 16- to 18-week-old spontaneously, hypertensive rats (SHR) compared with age-matched normotensive Wistar-Kyoto rats (WKY). Vmax is dependent on actomyosin ATPase activity, and this activity is in turn dependent on the level of phosphorylation of the 20-kDa myosin light chain (MLC20) normally a function of calcium concentration. In this article, methods are described and data are presented from studies addressing possible intracellular regulatory mechanisms that might lead to the altered contractility of the SHR arterial muscle. In one study, myofibrillar protein was extracted from 16- to 18-week-old SHR and WKY caudal arterial muscle. The Mg(2+)-activated ATPase activity was measured under conditions where the Ca2+ concentration was controlled. In another study, the amount of myosin present and relative proportions of the myosin heavy chain (MHC) isoforms were determined by quantitative SDS-PAGE using heavy molecular weight standards and bovine serum albumin as the standard for concentration. In a third study, MLC20 phosphorylation levels in electrically stimulated arterial muscle were determined by urea glycerol gel electrophoresis and Western blot analyses. The SHR (n = 6) myofibrillar ATPase liberated 0.011 +/- 0.003 mumol Pi/mg myosin/min, which was significantly more than the 0.006 +/- 0.001 mumol Pi/mg myosin/min liberated by the WKY (n = 4) myofibrillar ATPase (P < 0.05). Consistent with the increased ATPase activity, phosphorylation of MLC20 was increased by 2.8 times as much in the SHR compared with the WKY electrically stimulated arterial muscle. However, there was no difference in MHC isoform pattern in the SHR compared with the WKY arterial muscle in contrast to the findings of at least one other laboratory. This discrepancy is discussed. The data reviewed in this article lead to the conclusions that an increased actin-activated myosin ATPase activity and MLC20 phosphorylation are likely responsible for the increased velocity of shortening previously reported in SHR arterial muscle and the increased ATPase activity is not a function of an increased myosin content or of altered MHC isoform pattern in the SHR muscle.

Hypoxia-induced pulmonary arterial contraction appears to be dependent on myosin light chain phosphorylation.

The signal transduction pathway of hypoxic pulmonary arterial contraction has not been elucidated. Phosphorylation of the 20-kDa myosin light chain (MLC20) is thought to be essential for vascular muscle contraction. However, there are reports that smooth muscle will contract in response to nonphysiological stimuli such as phorbol esters without the involvement of MLC20 phosphorylation. The purpose of this study was to determine if hypoxia-induced pulmonary arterial contraction is dependent on MLC20 phosphorylation. Isolated rat pulmonary and carotid (for comparative purposes) arterial strips were contracted with 80 mM KCl to establish maximum active tension in response to membrane depolarization. The strips were then stimulated with one of the following: 30 mM KCl, 1 microM phenylephrine, 0.01 microM angiotensin II, 1 microM phorbol 12-myristate 13-acetate (PMA), or hypoxia (95% N2-5% CO2). In some experiments ML-9, a myosin light chain kinase inhibitor, or calphostin C, a protein kinase C (PKC) inhibitor, was introduced into the bath before hypoxia. Isometric tension was recorded as a function of time. Muscle strips were freeze-clamped (liquid N2) at various time points during the course of responses to the various stimuli. MLC20 phosphorylation levels were measured by ureaglycerol gel electrophoresis followed by Western blot procedure. Results show that increased MLC20 phosphorylation correlates with initiation of pulmonary arterial smooth muscle contraction in response to all agonists with the exception of PMA, a known activator of PKC. The MLC20 phosphorylation levels correlate with tension development in response to hypoxia, and ML-9 abolished the hypoxic contractions. In contrast, hypoxia relaxed carotid arterial muscle, and there was a corresponding decrease in the MLC20 phosphorylation level. In conclusion, hypoxia appears to result in MLC20 phosphorylation-mediated contraction in conduit pulmonary arterial muscle and in MLC20 dephosphorylation-mediated relaxation in systemic arterial muscle.

The vein utilizes different sources of energy than the artery during pulmonary hypoxic vasoconstriction.

Recent studies have shown that the contractile response to hypoxia is much greater in the pulmonary vein than in the artery. The purpose of this study was to investigate the effects of substrate utilization and oxidative phosphorylation on the responses of the pulmonary vein and artery to acute hypoxia. Isolated rat pulmonary arterial and venous rings were placed in tissue baths containing Earle's balanced salt solution (37 degrees C, 95% O2/5% CO2, pH 7.4), and attached to force transducers. The vascular rings were equilibrated for 1 h and then contracted maximally with 80 mM KCl to establish maximum active tension development (Po). Following washout and complete relaxation, the rings were incubated with the following substrates or metabolic inhibitors for 30-40 min: varying concentrations of glucose (0, 5.5, 10, or 20 mM), or glycolytic intermediates (4 mM pyruvate or 4 mM lactate), or inhibitors of glycolysis (50 mM 2-deoxyglucose or 0.1 mM iodoacetate), or an inhibitor of oxidative phosphorylation (0.1 microM rotenone). Vascular rings were then made hypoxic by lowering the bath Po2 to 30 torr. The pulmonary vein responded with a single contraction while the artery responded biphasically as previously reported. The pulmonary venous hypoxic response was not affected by the absence of glucose but was inhibited by high glucose concentrations. Neither glucose metabolic intermediates (pyruvate or lactate) nor the glycolysis inhibitor 2-deoxyglucose had any effect on the pulmonary venous response to hypoxia. However, inhibition of oxidative phosphorylation by rotenone inhibited the venous hypoxic response. In contrast, the pulmonary arterial phase 1 contraction to hypoxia was inhibited and phase 2 contraction was abolished in glucose-free solution. This effect was not due to the decreased production of glucose metabolic intermediates, since addition of pyruvate or lactate did not reverse the decreased arterial hypoxic response in glucose-free solution. Increasing the glucose concentration did not affect phase 1 contraction, but 20 mM glucose inhibited the phase 2 contraction. Inhibition of glycolysis with 2-deoxyglucose or iodoacetate decreased phase 1 contraction and abolished the phase 2 contraction. Inhibition of oxidative ATP production with rotenone abolished phase 1 but not phase 2 contraction. In conclusion, (1) the pulmonary venous response to hypoxia is unaffected by inhibition of glycolysis but is inhibited by high glucose and by inhibition of oxidative ATP production; (2) the pulmonary arterial hypoxic phase 1 contraction is dependent on oxidative ATP production; and (3) the phase 2 contraction of the pulmonary arterial hypoxic response depends on glycolytic ATP production but not on oxidative ATP production. These results indicate that the pulmonary vein and artery preferentially utilize different sources of energy for hypoxic contractions.

Chronic hypoxia impairs pulmonary venous smooth muscle contraction.

Chronic hypoxia increases total pulmonary vascular resistance and causes pulmonary hypertension. Although the effect of chronic hypoxia on pulmonary arterial tissue has been extensively studied, very little is known about the effects on the pulmonary vein. The purpose of the present investigation was to determine the effect of chronic hypoxia on pulmonary venous reactivity to several vasoactive agonists and on the venous response to acute hypoxia. Isolated pulmonary venous rings were taken from rats exposed to 2, 7, and 14 days of hypoxia (FIO2 = 0.1). A decrease in the response of the pulmonary vein to KCl was observed after 14 days of hypoxia. The reactivity (maximum active force produced) of the pulmonary vein in response to phenylephrine (PE) was reduced after 7 days of hypoxia. The response of the pulmonary vein to angiotensin II (AII) was more sensitive to the effects of chronic hypoxia since decreased reactivity to angiotensin II occurred after only 2 days of hypoxia. Prolonged hypoxia (14 days) had no further effect on the decreased reactivities to PE and AII. The sensitivities of pulmonary venous muscle to PE and AII were decreased (increased ED50 values) by 2 days of chronic hypoxia, but tended to return to control levels after 7 and 14 days of hypoxia. However, the contractile response of the pulmonary vein to acute hypoxia was not changed even after 14 days of chronic hypoxia. These results suggest that chronic hypoxia: (1) impairs pulmonary venous smooth muscle contractility; (2) reduces pulmonary venous reactivity and sensitivity to phenylephrine and angiotensin II; and (3) does not alter the pulmonary venous contractile response to acute hypoxia.

Changes in arterial smooth muscle contractility, contractile proteins, and arterial wall structure in spontaneous hypertension.

Heart disease, stroke, and kidney failure are leading causes of death. Essential hypertension is the major predisposing risk factor of cardiovascular disease. Yet, after several decades of intensive investigation, the initiating causative mechanism of essential hypertension is still unknown. However, investigators in the field generally agree that an increased total peripheral resistance (TPR) is the fundamental hemodynamic disorder in essential hypertension. This review addresses the hypothesis that the increased TPR of essential hypertension is due to a defective mechanism in the contractility of arterial smooth muscle. Force-velocity and length-tension studies have shown that both caudal arterial muscle and mesenteric resistance arterial muscle from spontaneously hypertensive rats (SHR) can shorten more and faster than muscle from normotensive control Wistar-Kyoto rats (WKY). In addition, the SHR muscle relaxation rate is slower compared with the WKY muscle. These alterations in mechanical behavior of SHR arterial muscle appear to be primary to the high blood pressure since MK-421 (enalapril maleate)-treated SHR arterial muscle shows the same increased velocity of shortening, increased shortening ability, and decreased relaxation rate as the untreated SHR muscle. MK-421 is an angiotensin-converting enzyme blocker. SHR maintained on MK-421 treatment have normal blood pressures in spite of being of the genetically hypertensive strain. While these findings are encouraging, several other important issues supporting the hypothesis require resolution and warrant review. Firstly, structural alterations of blood vessel walls in hypertension cause the walls to thicken and encroach on the vessel lumens contributing to the increased TPR. Whether such wall thickening is the cause or consequence of high blood pressure has been controversial in the literature. In this report, data are presented from a study in which MK-421-treated SHR were utilized as a model of prehypertensive SHR. Light micrograph observations and morphometric analyses were made of cross-sections of mesenteric resistance arteries from SHR, MK-421-treated SHR, and WKY. Results show that the MK-421-treated SHR resistance arteries had media thicknesses and a number of smooth muscle cell layers that were significantly less than in the untreated SHR and not different from the WKY. Secondly, velocity of shortening is dependent on actomyosin ATPase activity, and, since maximum velocity of shortening has been shown to be increased in SHR arterial muscle, it became necessary to know whether or not an increased actomyosin ATPase activity might be responsible. Therefore, data from a study of SHR and WKY caudal arterial myofibrillar ATPase activities are compared.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic strain stimulates dephosphorylation of the 20kDa regulatory myosin light chain in vascular smooth muscle cells.

The role of cyclic strain in the regulation of 20 kDa myosin light chain phosphorylation (MLC20) in cultured smooth muscle cells (SMC) is unknown. The objective of this study was to determine whether cyclic strain stimulates the dephosphorylation of MLC20 in serum-fed SMC displaying a high basal level of phosphorylation. Confluent bovine aortic SMC were subjected to 10% average strain at 60 cycles per minute for 30 and 60 minutes. Basal MLC20 phosphorylation (N = non,M = mono,D = di) of serum-fed SMC was as follows: N = 34%:M = 27%:D = 39%. After 60 min of cyclic strain, both mono and diphosphorylated MLC20 were decreased to 21 and 15% respectively. The strain-induced dephosphorylation of MLC20 was partially inhibited by the protein phosphatase 1/2A inhibitor, calyculin A (5 nM). However, phosphorylase a phosphatase activities in Triton-soluble and insoluble fractions of SMC were unaffected by cyclic strain. The data suggest that cyclic strain causes dephosphorylation of MLC20 in SMC which may be partially due to activation of MLC20 phosphatase and/or inhibition of MLC20 phosphorylation.

Contractility and myosin heavy chain isoform patterns in developing tracheal muscle.

Changes in airway smooth muscle reactivity with development may be caused by either modification of the excitation-contraction coupling system or alteration of the contractile apparatus. The mechanism responsible for the reported changes in reactivity was addressed in this study by examining airway smooth muscle contractility and myosin heavy chain isoform patterns as a function of post-neonatal development. Changes in length and force, in response to supramaximal electrical stimulation, were recorded simultaneously as functions of time for tracheal smooth muscle (TSM) strips from 8-week-old and 25-week-old male rabbits. Both the passive and active length-tension (L-T) curves as well as the force-velocity (F-V) curves for the two age groups of rabbit TSM were not significantly different indicating no changes in contractility during post-neonatal development in rabbits. This conclusion is surprising in light of reports of myosin heavy chain (MHC) isoform shifts in porcine trachealis during comparable periods of development. Therefore, MHC isoform ratios were compared by sodium dodecyl sulfate-polyacrylimide gel electrophoresis for tracheal smooth muscle from male rabbits of 8 and 25 weeks of age. Unlike the reported MHC isoform shifts in the pig tracheal muscle, the rabbit trachealis showed no difference in MHC isoform ratios between the two age groups compared in this study. In conclusion, no changes occur in contractility or MHC isoform patterns during post-neonatal development of rabbit tracheal smooth muscle. Therefore, reported changes in airway muscle reactivity are likely due to changes in receptors or in second messenger systems rather than to changes in the contractile apparatus.

Pulmonary arterial smooth muscle contractility in hypoxia-induced pulmonary hypertension.

The highly compliant low-resistance pulmonary vasculature is markedly altered with chronic hypoxia. Remodeling in response to hypoxia and/or hypertension involves hypertrophy and hyperplasia of smooth muscle and excessive deposition of connective tissue that likely contributes to the maintenance or exasperates the already elevated pulmonary arterial (PA) pressure. The purpose of this study was to investigate the effect of chronic hypoxia on the contractile properties of PA smooth muscle. Isometric and isotonic experiments were performed on excised PA rings from pulmonary hypertensive (induced by 14 days of hypoxia) Sprague-Dawley rats. A doubling of the vessel wall thickness occurred during the development of hypoxia-induced pulmonary hypertension. Functionally, there was a decrease in isometric stress (force to cross-sectional area ratio). No difference was detected in the velocity of shortening or in total shortening ability. This study provides evidence that, in addition to the morphological changes, changes in PA smooth muscle contractility also appear to play a role in the development and/or maintenance of hypoxia-induced pulmonary hypertension.

Neonatal hypoxia: long term effects on pulmonary arterial muscle.

The purpose of this study was to determine if neonatal hypoxia alters pulmonary arterial smooth muscle (PASM) function in young adult rats. One day old rats were made hypoxic (FIO2 = 0.1) for 5 days, then maintained under normoxic conditions until young adulthood (45-50 days). Age-matched rats were used as controls. Body weight, hematocrit, dry lung weight, and right to left heart ratios were measured. Reactivity and/or responsiveness of rings of main right and left pulmonary artery of the adult to various agonists, including high K+ (80 mM KCl), norepinephrine (NE), serotonin (5HT), adenosine (AD), and acute in vitro hypoxic vasoconstriction were assessed. Isometric force production was normalized to calculated tissue cross-sectional area (N/cm2). Maximum force production (PO) in response to 80 mM KCl for isolated rings from the hypoxic group was significantly less than for controls. Isometric force production in response to NE or to 5HT was also lower in the hypoxic group although the difference was significant for 5HT only when the endothelium was rendered non-functional. When the endothelium was intact, arterial rings from experimental animals relaxed at low doses of adenosine (10(-8) M to 10(-5) M), while control arterial muscle showed no response at these concentrations. The mean dose-response curve for NE from preparations with intact endothelium from experimental animals was significantly lower than that for the control animals, at least at doses greater than 10(-7) M. To mimic acute hypoxic pulmonary vasoconstriction, isolated rings of the main right and left pulmonary artery were precontracted with either 30 mM KCl or 2.5 x 10(-7) M NE and then made hypoxic by lowering muscle bath PO2 to 30-40 mmHg. In conclusion there was no difference in the hypoxic response per se between arterial rings from experimental animals and controls. However, maximum reactivity to high potassium stimulation and to norepinephrine stimulation is decreased in pulmonary arterial smooth muscle of adult animals that had been exposed to 5 days of hypoxia as neonates.

Pulmonary vein contracts in response to hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important regulatory mechanism in matching regional blood flow and ventilation. The HPV response has been well documented on the arterial side, but the response of pulmonary veins to hypoxia has received little attention. The purpose of the present study was to determine whether isolated rat pulmonary veins contract in response to decreased PO2 and, if so, to compare the venous response with that of the pulmonary artery. Rat pulmonary venous and arterial rings were attached to force transducers and precontracted with either a submaximal dose of KCl or norepinephrine under normoxic conditions and then made hypoxic. The pulmonary venous hypoxic response consisted of a single sustained contraction, whereas the arterial response to hypoxia was biphasic, consisting of an initial rapid contraction and then a slowly developed but sustained contraction. The venous hypoxic contraction was significantly greater in magnitude than either phase 1 or phase 2 of the arterial response. Endothelium denudation did not affect the venous hypoxic response. However, the venous hypoxic response was dependent on the level of precontractile tone and also appeared to be dependent on the specific contractile agonist. Unlike the isolated arterial phase 1 hypoxic response (but similar to the arterial phase 2 response) the pulmonary venous hypoxic contraction was inhibited in Ca(2+)-free media or by Ca2+ channel blockers. In summary, pulmonary venous smooth muscle contracts to a relatively greater degree in response to severe hypoxia than does pulmonary arterial smooth muscle. The venous hypoxic response is endothelium independent, as is phase 2 of the arterial response.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol trisphosphate is involved in norepinephrine- but not in hypoxia-induced pulmonary arterial contraction.

The role that second messengers play in pulmonary vasoconstriction is not understood. The purpose of this study was to directly measure inositol phosphates in isolated pulmonary arterial preparations before and during norepinephrine (NE) stimulation and acute hypoxia. Rat main pulmonary arteries were isolated and incubated with myo-[3H]-inositol. After incubation, control tissue was stimulated with 0.5 microM NE or 30 mM KCl. Test preparations were precontracted with 30 mM KCl and then exposed to hypoxia. Samples were homogenized and applied to a high-pressure liquid chromatography column for analysis of inositol phosphates. Results show that inositol trisphosphate (IP3) increases twofold at 5 s following NE stimulation. Thirty micromolars of KCl results in a slight but significant increase in IP3 formation at 5 min following the stimulation. Phentolamine inhibits the KCl-induced increase in IP3 formation, whereas A23187 has no effect on IP3 levels. Hypoxia caused a biphasic contraction in the precontracted isolated rat pulmonary artery. IP3 levels did not change during the hypoxic period. In conclusion, NE causes a rapid increase in IP3 formation consistent with the time course of production of an excitation-contraction coupling second messenger. However, inositol trisphosphate is not involved in the signal transduction pathway leading to pulmonary arterial contraction induced by hypoxia.

Pulmonary arterial hypoxic contraction: signal transduction.

The response of isolated rat pulmonary arteries to acute hypoxia has previously been reported to be biphasic, consisting of an initial rapid contraction of short duration, followed by partial relaxation (phase 1) and then a second slowly developed but sustained contraction (phase 2). The purpose of this study was to determine the following: 1) whether products from the endothelium might be required, 2) whether extra- and/or intracellular calcium or protein kinase C might be second messengers in mediating the pulmonary arterial hypoxic contraction, and 3) whether or not guanosine 3',5'-cyclic monophosphate (cGMP), endothelium-derived relaxing factor (EDRF), prostaglandin I2 (PGI2) or A2 adenosine receptor activation is involved in phase 1 relaxation. Neither Ca(2+)-free media nor verapamil (a Ca2+ channel blocker) altered the phase 1 contraction, but the phase 2 contraction was abolished by either of these treatments. Ryanodine (a sarcoplasmic reticulum Ca2+ depleter) had no effect on phase 1 contraction. H-7 (a PKC inhibitor) inhibited the phase 2 contraction, whereas it had no effect on phase 1 contraction. Removal of the endothelium abolished phase 1 contraction in either Ca(2+)-free media or normal Ca2+ media but did not alter phase 2 contraction or phase 1 relaxation. Neither methylene blue (guanylate cyclase inhibitor), N omega-nitro-L-arginine, (EDRF blocker), acetylsalicylic acid (cyclooxygenase inhibitor), xanthine amino congener (adenosine receptor blocker), nor glybenclamide blocked the phase 1 relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated neutrophils alter contractile properties of the pulmonary artery.

Activated neutrophils produce a wide array of products (free radicals, arachidonate metabolites, degradative enzymes), cause hemodynamic effects and increased permeability in isolated blood-free perfused lungs, and evoke direct injury to cultured endothelial cells. The aims of this study were to investigate the response of isolated rat pulmonary arterial rings to activated neutrophils, the role of intact endothelium in these responses, and which neutrophil products were responsible for the observed effects. Neutrophils activated with phorbol myristate acetate caused an initial increase in tension and a subsequent decreased recovery contraction to KCl. Neutrophils activated with formylmethionylleucylphenylalanine also caused an increase in tension but did not result in decreased recovery, suggesting different mechanisms for these two effects. The contractile response was dependent on endothelium, whereas the decline in recovery still occurred in the absence of endothelium. Filtrate from activated neutrophils did not cause the contractile response, but recovery was decreased. Neither addition of catalase + superoxide dismutase nor decreased superoxide release due to prior activation of neutrophils altered the initial contraction or the decline in recovery contractile ability, suggesting that oxygen free radical products were not responsible for either effect. The cyclooxygenase inhibitors (ibuprofen and indomethacin), the thromboxane A2 synthetase inhibitor (OKY-046), and pretreatment of the neutrophils with aspirin inhibited the contractile response but did not prevent the decrease in recovery. A mixture of antiproteases did not protect the arterial muscle from the decline in recovery. Although cyclooxygenase products may be involved in initiating the contraction in response to activated neutrophils, the mechanism resulting in subsequent loss of force-developing ability is unclear.

Laser and suture anastomosis: passive compliance and active force production.

One carotid from each dog underwent a laser anastomosis and the other carotid an interrupted suture repair. One or eight weeks later (n greater than or equal to 4 dogs/time period); four rings (1 mm in length) containing the laser or suture anastomosis or the normal artery (two/dog) were removed. Using a photoelectric force transducer and lever system, the ring was stretched in increments and passive force measured. At each length, the arterial muscle was stimulated and active force measured. The mean laser and control passive length/tension (L/T) curves were not different (P greater than 0.05), but the suture curve was shifted downward (P less than 0.05). The mean laser and suture active L/T curves were similar at 1 week (greater than 0.05) and lower than the control curve (P less than 0.04). At 8 weeks, the laser-repaired vessels produced an active force similar to control muscle (P greater than 0.05) but the suture repairs could not generate this active force (P less than 0.05). These data suggest that the laser repair and normal artery are more mechanically compatible than the suture repair as studied by this method.